Treatment of psychological trauma

ABSTRACT

The present specification discloses methods of treating a psychological trauma in an individual using botulinum toxins and/or a Targeted Exocytosis Modulator, and compositions thereof.

This application is a Continuation Application of U.S. application Ser.No. 13/495,605, filed Jun. 13, 2012, which claims the benefit ofpriority pursuant to 35 U.S.C. §119(e) to U.S. provisional patentapplications Serial No. 61/496,192, filed Jun. 13, 2011 and incorporatedentirely by reference.

Psychological trauma is a type of damage to the psyche that occurs as aresult of a traumatic event. A traumatic event involves a singleexperience, or an enduring or repeating event or events, that completelyoverwhelm the individual's ability to cope or integrate the ideas andemotions involved with that experience. The sense of being overwhelmedcan be delayed by weeks, years or even decades, as the person strugglesto cope with the immediate circumstances. intense feelings of anger maysurface frequently, sometimes in very inappropriate or unexpectedsituations, as danger may always seem to be present. Upsetting memoriessuch as images, thoughts, or flashbacks may haunt the person, andnightmares may be frequent. Insomnia may occur as lurking fears andinsecurity keep the person vigilant and on the lookout for danger, bothday and night.

Post-traumatic stress disorder (PTSD) is a severe anxiety disorder thatcan develop after exposure to any event that results in psychologicaltrauma. A common example of an event triggering PTSD is the repeatedthreat of death to oneself or a companion, such as during combatoperations. Many soldiers experience PTSD after returning from a war.Other examples of events leading to PTSD include threats or assaults onthe physical, sexual, or psychological integrity to oneself or acompanion. The psychological trauma overwhelms the individual's abilityto cope. PTSD may be more likely to be caused by physical orpsychological trauma caused by humans such as rape, war, or terroristattack than trauma caused by natural disasters. Symptoms for PTSDinclude re-experiencing the original trauma(s) through flashbacks ornightmares, avoidance of stimuli associated with the trauma, andincreased arousal—such as difficulty falling or staying asleep, anger,and hyper-vigilance. Findings indicate that a failure to provideadequate treatment to children after they suffer a traumatic experience,depending on their vulnerability and the severity of the trauma, willultimately lead to PTSD symptoms in adulthood. PTSD symptoms may resultwhen a traumatic event causes an overactive adrenaline response, whichcreates deep neurological patterns in the brain. These patterns canpersist long after the event that triggered the fear, making anindividual hyper-responsive to future fearful situations.

One of the first descriptions of PTSD was made by the Greek historianHerodotus in 490 BCE. He described, during the Battle of Marathon, anAthenian soldier who suffered no injury from war but became permanentlyblind after witnessing the death of a fellow soldier. Previous diagnosesnow considered historical equivalents of PTSD include railway spine,stress syndrome, shell shock, battle fatigue, or traumatic war neurosis.Even after 2,500 years, while a variety of medications have shownadjunctive benefit in reducing PTSD symptoms, there is no clear drugtreatment for PTSD.

Complex post-traumatic stress disorder (C-PTSD) is a psychologicalinjury that results from protracted exposure to prolonged social and/orinterpersonal trauma with lack or loss of control, disempowerment, andin the context of either captivity or entrapment. C-PTSD is distinctfrom, but similar to, PTSD. The present invention includes the treatmentof C-PTSD.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show a schematic of the current paradigm ofneurotransmitter release and Clostridial toxin intoxication in a centraland peripheral neuron. FIG. 1A shows a schematic for theneurotransmitter release mechanism of a central and peripheral neuron.The release process can be described as comprising two steps: 1) vesicledocking, where the vesicle-bound SNARE protein of a vesicle containingneurotransmitter molecules associates with the membrane-bound SNAREproteins located at the plasma membrane; and 2) neurotransmitterrelease, where the vesicle fuses with the plasma membrane and theneurotransmitter molecules are exocytosed. FIG. 1B shows a schematic ofthe intoxication mechanism for tetanus and botulinum toxin activity in acentral and peripheral neuron. This intoxication process can bedescribed as comprising four steps: 1) receptor binding, where aClostridial toxin binds to a Clostridial receptor system and initiatesthe intoxication process; 2) complex internalization, where after toxinbinding, a vesicle containing the toxin/receptor system complex isendocytosed into the cell; 3) light chain translocation, where multipleevents are thought to occur, including, e.g., changes in the internal pHof the vesicle, formation of a channel pore comprising the HN domain ofthe Clostridial toxin heavy chain, separation of the Clostridial toxinlight chain from the heavy chain, and release of the active light chainand 4) enzymatic target modification, where the activate light chain ofClostridial toxin proteolytically cleaves its target SNARE substrate,such as, e.g., SNAP-25, VAMP or Syntaxin, thereby preventing vesicledocking and neurotransmitter release.

FIG. 2 shows the domain organization of naturally-occurring Clostridialtoxins. The single-chain form depicts the amino to carboxyl linearorganization comprising an enzymatic domain, a translocation domain, anda retargeted peptide binding domain. The di-chain loop region locatedbetween the translocation and enzymatic domains is depicted by thedouble SS bracket. This region comprises an endogenous di-chain loopprotease cleavage site that upon proteolytic cleavage with anaturally-occurring protease, such as, e.g., an endogenous Clostridialtoxin protease or a naturally-occurring protease produced in theenvironment, converts the single-chain form of the toxin into thedi-chain form. Above the single-chain form, the H_(CC) region of theClostridial toxin binding domain is depicted. This region comprises theβ-trefoil domain which comprises in an amino to carboxyl linearorganization an α-fold, a β4/β5 hairpin turn, a β-fold, a β8/β9 hairpinturn and a γ-fold.

FIGS. 3A and 3B show Targeted Exocytosis Modulator (TEM) domainorganization with a targeting domain located at the amino terminus of aTEM. FIG. 3A depicts the single-chain polypeptide form of a TEM with anamino to carboxyl linear organization comprising a targeting domain, atranslocation domain, a di-chain loop region comprising an exogenousprotease cleavage site (P), and an enzymatic domain. Upon proteolyticcleavage with a P protease, the single-chain form of the TEM isconverted to the di-chain form. FIG. 3B depicts the single polypeptideform of a TEM with an amino to carboxyl linear organization comprising atargeting domain, an enzymatic domain, a di-chain loop region comprisingan exogenous protease cleavage site (P), and a translocation domain.Upon proteolytic cleavage with a P protease, the single-chain form ofthe TEM is converted to the di-chain form.

FIGS. 4A, 4B, 4C and 4D show a TEM domain organization with a targetingdomain located between the other two domains. FIG. 4A depicts the singlepolypeptide form of a TEM with an amino to carboxyl linear organizationcomprising an enzymatic domain, a di-chain loop region comprising anexogenous protease cleavage site (P), a targeting domain, and atranslocation domain. Upon proteolytic cleavage with a P protease, thesingle-chain form of the TEM is converted to the di-chain form. FIG. 4Bdepicts the single polypeptide form of a TEM with an amino to carboxyllinear organization comprising a translocation domain, a di-chain loopregion comprising an exogenous protease cleavage site (P), a targetingdomain, and an enzymatic domain. Upon proteolytic cleavage with a Pprotease, the single-chain form of the TEM is converted to the di-chainform. FIG. 4C depicts the single polypeptide form of a TEM with an aminoto carboxyl linear organization comprising an enzymatic domain, atargeting domain, a di-chain loop region comprising an exogenousprotease cleavage site (P), and a translocation domain. Upon proteolyticcleavage with a P protease, the single-chain form of the TEM isconverted to the di-chain form. FIG. 4D depicts the single polypeptideform of a TEM with an amino to carboxyl linear organization comprising atranslocation domain, a targeting domain, a di-chain loop regioncomprising an exogenous protease cleavage site (P), and an enzymaticdomain. Upon proteolytic cleavage with a P protease, the single-chainform of the TEM is converted to the di-chain form.

FIGS. 5A and 5B show a TEM domain organization with a targeting domainlocated at the carboxyl terminus of the TEM. FIG. 5A depicts the singlepolypeptide form of a TEM with an amino to carboxyl linear organizationcomprising an enzymatic domain, a di-chain loop region comprising anexogenous protease cleavage site (P), a translocation domain, and atargeting domain. Upon proteolytic cleavage with a P protease, thesingle-chain form of the TEM is converted to the di-chain form. FIG. 5Bdepicts the single polypeptide form of a TEM with an amino to carboxyllinear organization comprising a translocation domain, a di-chain loopregion comprising an exogenous protease cleavage site (P), an enzymaticdomain, and a targeting domain. Upon proteolytic cleavage with a Pprotease, the single-chain form of the TEM is converted to the di-chainform.

DESCRIPTION

For purposes of this application, any BoNT units are given in terms ofBotox® units. Therapeutically effective units of other BoNT products,such as Xeomin® and Dysport®, may be titrated by one of ordinary skillin the art.

The ability of Clostridial toxins, such as, e.g., Botulinum neurotoxins(BoNTs), BoNT/A, BoNT/B, BoNT/C1, BoNT/D, BoNT/E, BoNT/F and BoNT/G, andTetanus neurotoxin (TeNT), to inhibit neuronal transmission are beingexploited in a wide variety of therapeutic and cosmetic applications,see e.g., William J. Lipham, COSMETIC AND CLINICAL APPLICATIONS OFBOTULINUM TOXIN (Slack, ed., 2004). Clostridial toxins commerciallyavailable as pharmaceutical compositions include, BoNT/A preparations,such as, e.g., BOTOX® (Allergan, Inc., Irvine, Calif. ),DYSPORT®/RELOXIN®, (Beaufour Ipsen, Porton Down, England), NEURONOX®(Medy-Tox, Inc., Ochang-myeon, South Korea), BTX-A (Lanzhou InstituteBiological Products, China) and XEOMIN® (Merz Pharmaceuticals, GmbH.,Frankfurt, Germany); and BoNT/B preparations, such as, e.g.,MYOBLOC™/NEUROBLOC™ (Solstice Neurosciences, Inc., South San Francisco,Calif.). As an example, BOTOX® is currently approved in one or morecountries for the following indications: achalasia, adult spasticity,anal fissure, back pain, blepharospasm, bruxism, cervical dystonia,essential tremor, glabellar lines or hyperkinetic facial lines,headache, hemifacial spasm, hyperactivity of bladder, hyperhidrosis,juvenile cerebral palsy, multiple sclerosis, myoclonic disorders, nasallabial lines, spasmodic dysphonia, strabismus and VII nerve disorder.See also, U.S. Pat. No. 5,714,468 (migraine) issued Feb. 3, 1998;Published U.S. Patent Application No. 2005019132 (headache), Ser. No.11/039,506, filed Jan. 18, 2005; Published U.S. Patent Application No.20050191320 (medication overuse headache), Ser. No. 10/789,180, filedFeb. 26, 2004; and U.S. Pat. No. 7,811,587 (neuropsychiatric disorders),issued Oct. 12, 2010; U.S. application Ser. No. 13/053,583, filed Mar.22, 2011 (depression); and, U.S. application Ser. No. 13/075,485, filedMar. 30, 2011 (migraine treatment), all incorporated entirely byreference.

Clostridial toxin therapies have been successfully used for manyindications. However, toxin administration in some applications can bechallenging because of the larger doses required to achieve a beneficialeffect. Larger doses can increase the likelihood that the toxin may movethrough the interstitial fluids and the circulatory systems, such as,e.g., the cardiovascular system and the lymphatic system, of the body,resulting in the undesirable dispersal of the toxin to areas nottargeted for toxin treatment. Such dispersal can lead to undesirableside effects, such as, e.g., inhibition of neurotransmitter release inneurons not targeted for treatment or paralysis of a muscle not targetedfor treatment. For example, a individual administered a therapeuticallyeffective amount of a BoNT/A treatment into the neck muscles forcervical dystonia may develop dysphagia because of dispersal of thetoxin into the oropharynx. As another example, a individual administereda therapeutically effective amount of a BoNT/A treatment into thebladder for overactive bladder may develop dry mouth and/or dry eyes.Thus, there still remains a need for treatments having the therapeuticeffects that only larger doses of a Clostridial toxin can currentlyprovide, but reduce or prevent the undesirable side-effects associatedwith larger doses of a Clostridial toxin administration.

Clostridia toxins produced by Clostridium botulinum, Clostridium tetani,Clostridium baratii and Clostridium butyricum are the most widely usedin therapeutic and cosmetic treatments of humans and other mammals.Strains of C. botulinum produce seven antigenically-distinct types ofBotulinum toxins (BoNTs), which have been identified by investigatingbotulism outbreaks in man (BoNT/A, BoNT/B, BoNT/E and BoNT/F), animals(BoNT/C1 and BoNT/D), or isolated from soil (BoNT/G). BoNTs possessapproximately 35% amino acid identity with each other and share the samefunctional domain organization and overall structural architecture. Itis recognized by those of skill in the art that within each type ofClostridial toxin there can be subtypes that differ somewhat in theiramino acid sequence, and also in the nucleic acids encoding theseproteins. For example, there are presently five BoNT/A subtypes,BoNT/A1, BoNT/A2, BoNT/A3 BoNT/A4 and BoNT/A5, with specific subtypesshowing approximately 89% amino acid identity when compared to anotherBoNT/A subtype. While all seven BoNT serotypes have similar structureand pharmacological properties, each also displays heterogeneousbacteriological characteristics. In contrast, tetanus toxin (TeNT) isproduced by a uniform group of C. tetani. Two other Clostridia species,C. baratii and C. butyricum, produce toxins, BaNT and BuNT, which arefunctionally similar to BoNT/F and BoNT/E, respectively.

A Clostridial toxin treatment inhibits neurotransmitter release bydisrupting the exocytotic process used to secret the neurotransmitterinto the synaptic cleft. There is a great desire by the pharmaceuticalindustry to expand the use of Clostridial toxin therapies beyond itscurrent myorelaxant applications to treat sensory, sympathetic, and/orparasympathetic nerve-based ailments, such as, e.g., various kinds ofsmooth muscle-based disorders. One approach that is currently beingexploited involves modifying a Clostridial toxin such that the modifiedtoxin has an altered cell targeting capability for a neuronal ornon-neuronal cell of interest. Called re-targeted endopeptidases orTargeted Vesicular Exocytosis Modulator Proteins (TVEMPs) or TargetedExocytosis Modulators (TEMs), these molecules achieve their exocytosisinhibitory effects by targeting a receptor present on the neuronal ornon-neuronal target cell of interest. This re-targeted capability isachieved by replacing the naturally-occurring binding domain of aClostridial toxin with a targeting domain showing a selective bindingactivity for a non-Clostridial toxin receptor present in a cell ofinterest. Such modifications to the binding domain result in a moleculethat is able to selectively bind to a non-Clostridial toxin receptorpresent on the target cell. A re-targeted endopeptidase can bind to atarget receptor, translocate into the cytoplasm, and exert itsproteolytic effect on the SNARE complex of the neuronal or non-neuronaltarget cell of interest.

The present specification discloses methods for treating an individualsuffering from physiological trauma disorders. This is accomplished byadministering a therapeutically effective amount of a compositioncomprising a Botulinum toxin (BoNT) and/or a TEM to an individual inneed thereof. The disclosed methods provide a safe outpatient-basedtreatment.

With reference to psychological trauma disorders as disclosed herein,and without wishing to be limited by any particular theory, it isbelieved that sympathetic, parasympathetic, and/or sensory neurons haveimportant functions in aspects of psychological trauma regulation andthat improper innervations from these types of neurons can contribute toone or more different types of psychological trauma disorders. As such,TEMs comprising a targeting domain for a receptor present onsympathetic, parasympathetic, and/or sensory neurons can reduce orprevent these improper innervations, thereby reducing or preventing oneor more symptoms associate with a psychological trauma disorder. It isfurther theorized that such a TEM in combination with a Clostridialtoxin can provide enhanced, if not synergistic, therapeutic benefitbecause such a combination also inhibit motor neurons. However, using acombination therapy of such a TEM with a Clostridial toxin, also allowsa lower dose of a Clostridial toxin to be administered to treat apsychological trauma disorder. This will result in a decrease in muscleweakness generated in the compensatory muscles relative to the currenttreatment paradigm. As such, a combined therapy using a Clostridialtoxin and a TEM comprising a targeting domain for a receptor present onsympathetic, parasympathetic, and/or sensory neurons can reduce orprevent these improper innervations, and in combination can reduce orprevent one or more symptoms associate with a psychological traumadisorder.

Thus, aspects of the present specification disclose methods of treatinga psychological trauma disorder in an individual, the methods comprisingthe step of administering to the individual in need thereof atherapeutically effective amount of a composition including a TEM,wherein administration of the composition reduces a symptom of thepsychological trauma disorder, thereby treating the individual. In someaspects, a TEM may comprise a targeting domain, a Clostridial toxintranslocation domain and a Clostridial toxin enzymatic domain. In someaspects, a TEM may comprise a targeting domain, a Clostridial toxintranslocation domain, a Clostridial toxin enzymatic domain, and anexogenous protease cleavage site. A targeting domain includes, withoutlimitation, a sensory neuron targeting domain, a sympathetic neurontargeting domain, or a parasympathetic neuron targeting domain.

Other aspects of the present specification disclose uses of a TEMdisclosed herein in the manufacturing a medicament for treating apsychological trauma disorder disclosed herein in an individual in needthereof.

Yet other aspects of the present specification uses of a TEM disclosedherein in the treatment of a psychological trauma disorder disclosedherein in an individual in need thereof.

Other aspects of the present specification disclose methods of treatinga psychological trauma disorder in an individual, the methods comprisingthe step of administering to the individual in need thereof atherapeutically effective amount of a composition including aClostridial neurotoxin and a TEM, wherein administration of thecomposition reduces a symptom of the psychological trauma, therebytreating the individual. A Clostridial neurotoxin includes, withoutlimitation, a Botulinum toxin (BoNT), a Tetanus toxin (TeNT), a Baratiitoxin (BaNT), and a Butyricum toxin (BuNT). In some aspects, a TEM maycomprise a targeting domain, a Clostridial toxin translocation domainand a Clostridial toxin enzymatic domain. In some aspects, a TEM maycomprise a targeting domain, a Clostridial toxin translocation domain, aClostridial toxin enzymatic domain, and an exogenous protease cleavagesite. A targeting domain includes, without limitation, a sensory neurontargeting domain, a sympathetic neuron targeting domain, or aparasympathetic neuron targeting domain.

Other aspects of the present specification disclose uses of aClostridial neurotoxin and a TEM disclosed herein in the manufacturing amedicament for treating a psychological trauma disorder disclosed hereinin an individual in need thereof.

Yet other aspects of the present specification uses of a Clostridialneurotoxin and a TEM disclosed herein in the treatment of apsychological trauma disorder disclosed herein in an individual in needthereof.

As used herein, “about” means approximately or nearly and in the contextof a numerical value or range set forth means ±15% of the numericalvalue range recited.

As used herein, “neurotoxin” means a biologically active molecule with aspecific affinity for a cell surface receptor of motor neurons (alsoknown as efferent or effector neurons). Neurotoxin includes Clostridialtoxins, preferably Clostridial botulinum toxins, both as non-complexedtoxin (having a molecular weight of about 150 kDa) and as complexed withone or more non-toxin, toxin associated proteins; the complexes havingmolecular weights of about 900 kD, 700, kD, 500 kD or 300 kD, forexample. Botulinum toxins can include toxins that are recombinantly madeand modified in accordance with known molecular techniques, that is, amodified neurotoxin means a neurotoxin which has had one or more of itsamino acids deleted, modified or replaced (as compared to the nativeneurotoxin) and includes neurotoxins made by recombinant technology aswell as variants and fragments of a native or recombinantly producedneurotoxin.

As used herein “treating” or “to treat” means to alleviate, modulate, oreliminate either a symptom of a condition or disorder or the conditionor disorder itself.

As used herein “local administration” or “locally administering” meansdirect administration of a pharmaceutical at, or to the vicinity of, asite on or within an animal body, at which site a biological effect ofthe pharmaceutical is desired. One example of local administration caninclude direct injection of a botulinum toxin. Topical administration asutilized herein is a type of local administration in which apharmaceutical agent is administered to a person's periclitoral area,such as for example to the periclitoral area to which botulinum toxin,for example, is to be administered in accordance with the teachingspresented herein.

As used herein “therapeutically effective” means an amount of toxinadministered that will reduce or ameliorate a condition or symptom (infrequency and/or intensity) in a subject. The therapeutically effectiveamount of toxin, such as a botulinum neurotoxin, delivered to a subject,is an amount that achieves a desired effect yet does not result inundesirable systemic side effects associated with systemic neurotoxinpoisoning, as known by those of ordinary skill in the art.

The combination of botulinum toxins and TEMs allows for dose reductionof active agents (with associated reduction in side effects) as well aspossible synergistic effects. Non-paralytic effects, and also possibleprophylactic effects especially when used early in the condition canprovide further benefits.

Clostridial toxins are released by Clostridial bacterium as complexescomprising the approximately 150-kDa Clostridial toxin along withassociated non-toxin proteins (NAPs). Identified NAPs include proteinspossessing hemagglutination activity, such, e.g., a hemagglutinin ofapproximately 17-kDa (HA-17), a hemagglutinin of approximately 33-kDa(HA-33) and a hemagglutinin of approximately 70-kDa (HA-70); as well asnon-toxic non-hemagglutinin (NTNH), a protein of approximately 130-kDa.Thus, the botulinum toxin type A complex can be produced by Clostridialbacterium as 900-kDa, 500-kDa and 300-kDa forms. Botulinum toxin types Band C₁ are apparently produced as only a 500-kDa complex. Botulinumtoxin type D is produced as both 300-kDa and 500-kDa complexes. Finally,botulinum toxin types E and F are produced as only approximately 300-kDacomplexes. The differences in molecular weight for the complexes are dueto differing ratios of NAPs. The toxin complex is important for theintoxication process because it provides protection from adverseenvironmental conditions, resistance to protease digestion, and appearsto facilitate internalization and activation of the toxin.

A Clostridial toxin itself is translated as a single chain polypeptidethat is subsequently cleaved by proteolytic scission within a disulfideloop by a naturally-occurring protease (FIG. 1). This cleavage occurswithin the discrete di-chain loop region created between two cysteineresidues that form a disulfide bridge. This post-translationalprocessing yields a di-chain molecule comprising an approximately 50 kDalight chain (LC) and an approximately 100 kDa heavy chain (HC) heldtogether by the single disulfide bond and non-covalent interactionsbetween the two chains. The naturally-occurring protease used to convertthe single chain molecule into the di-chain is currently not known. Insome serotypes, such as, e.g., BoNT/A, the naturally- occurring proteaseis produced endogenously by the bacteria serotype and cleavage occurswithin the cell before the toxin is release into the environment.However, in other serotypes, such as, e.g., BoNT/E, the bacterial strainappears not to produce an endogenous protease capable of converting thesingle chain form of the toxin into the di-chain form. In thesesituations, the toxin is released from the cell as a single-chain toxinwhich is subsequently converted into the di-chain form by anaturally-occurring protease found in the environment.

Each mature di-chain molecule of a Clostridial toxin comprises threefunctionally distinct domains: 1) an enzymatic domain located in thelight chain (LC) that includes a metalloprotease region containing azinc-dependent endopeptidase activity which specifically targets corecomponents of the neurotransmitter release apparatus; 2) a translocationdomain contained within the amino-terminal half of the heavy chain(H_(N)) that facilitates release of the LC from intracellular vesiclesinto the cytoplasm of the target cell; and 3) a binding domain foundwithin the carboxyl-terminal half of the heavy chain (H_(C)) thatdetermines the binding activity and binding specificity of the toxin tothe receptor complex located at the surface of the target cell. TheH_(C) domain comprises two distinct structural features of roughly equalsize that indicate function and are designated the H_(CN) and H_(CC)subdomains.

Clostridial toxins act on the nervous system by blocking the release ofacetylcholine (ACh) at the pre-synaptic neuromuscular junction. Thebinding, translocation and enzymatic activity of these three functionaldomains are all necessary for toxicity. While all details of thisprocess are not yet precisely known, the overall cellular intoxicationmechanism whereby Clostridial toxins enter a neuron and inhibitneurotransmitter release is similar, regardless of serotype or subtype.Although applicants have no wish to be limited by the followingdescription, the intoxication mechanism can be described as comprisingat least four steps: 1) receptor binding, 2) complex internalization, 3)light chain translocation, and 4) enzymatic target modification (FIG.1). The process is initiated when the binding domain of a Clostridialtoxin binds to a toxin-specific receptor system located on the plasmamembrane surface of a target cell. The binding specificity of a receptorcomplex is thought to be achieved, in part, by specific combinations ofgangliosides and protein receptors that appear to distinctly compriseeach Clostridial toxin receptor complex. Once bound, the toxin/receptorcomplexes are internalized by endocytosis and the internalized vesiclesare sorted to specific intracellular routes. The translocation stepappears to be triggered by the acidification of the vesicle compartment.This process seems to initiate pH-dependent structural rearrangementsthat increase hydrophobicity, create a pore in the vesicle membrane, andpromote formation of the di-chain form of the toxin. Once di-chainformation occurs, light chain endopeptidase of the toxin is releasedfrom the intracellular vesicle via the pore into the cytosol where itappears to specifically target one of three known core components of theneurotransmitter release apparatus. These core proteins,vesicle-associated membrane protein (VAMP)/synaptobrevin, synaptosomal-associated protein of 25 kDa (SNAP-25) and Syntaxin, are necessary forsynaptic vesicle docking and fusion at the nerve terminal and constitutemembers of the soluble N-ethylmaleimide-sensitive factor-attachmentprotein-receptor (SNARE) family. BoNT/A and BoNT/E cleave SNAP-25 in thecarboxyl-terminal region, releasing a nine or twenty-six amino acidsegment, respectively, and BoNT/C1 also cleaves SNAP-25 near thecarboxyl-terminus. The botulinum serotypes BoNT/B, BoNT/D, BoNT/F andBoNT/G, and tetanus toxin, act on the conserved central portion of VAMP,and release the amino-terminal portion of VAMP into the cytosol. BoNT/C1cleaves syntaxin at a single site near the cytosolic membrane surface.

Aspects of the present specification disclose, in part, in part, aClostridial toxin. As used herein, the term “Clostridial toxin” refersto any toxin produced by a Clostridial toxin strain that can execute theoverall cellular mechanism whereby a Clostridial toxin intoxicates acell and encompasses the binding of a Clostridial toxin to a low or highaffinity Clostridial toxin receptor, the internalization of thetoxin/receptor complex, the translocation of the Clostridial toxin lightchain into the cytoplasm and the enzymatic modification of a Clostridialtoxin substrate. Non-limiting examples of Clostridial toxins include aBotulinum toxin like BoNT/A, a BoNT/B, a BoNT/C₁, a BoNT/D, a BoNT/E, aBoNT/F, a BoNT/G, a Tetanus toxin (TeNT), a Baratii toxin (BaNT), and aButyricum toxin (BuNT). The BoNT/C₂ cytotoxin and BoNT/C₃ cytotoxin, notbeing neurotoxins, are excluded from the term “Clostridial toxin.” AClostridial toxin disclosed herein includes, without limitation,naturally occurring Clostridial toxin variants, such as, e.g.,Clostridial toxin isoforms and Clostridial toxin subtypes; non-naturallyoccurring Clostridial toxin variants, such as, e.g., conservativeClostridial toxin variants, non-conservative Clostridial toxin variants,Clostridial toxin chimeric variants and active Clostridial toxinfragments thereof, or any combination thereof.

A Clostridial toxin disclosed herein also includes a Clostridial toxincomplex. As used herein, the term “Clostridial toxin complex” refers toa complex comprising a Clostridial toxin and non-toxin associatedproteins (NAPs), such as, e.g., a Botulinum toxin complex, a Tetanustoxin complex, a Baratii toxin complex, and a Butyricum toxin complex.Non-limiting examples of Clostridial toxin complexes include thoseproduced by a Clostridium botulinum, such as, e.g., a 900-kDa BoNT/Acomplex, a 500-kDa BoNT/A complex, a 300-kDa BoNT/A complex, a 500-kDaBoNT/B complex, a 500-kDa BoNT/C₁ complex, a 500-kDa BoNT/D complex, a300-kDa BoNT/D complex, a 300-kDa BoNT/E complex, and a 300-kDa BoNT/Fcomplex.

Clostridial toxins can be produced using standard purification orrecombinant biology techniques known to those skilled in the art. See,e.g., Hui Xiang et al., Animal Product Free System and Process forPurifying a Botulinum Toxin, U.S. Pat. No. 7,354,740, which is herebyincorporated by reference in its entirety. For example, a BoNT/A complexcan be isolated and purified from an anaerobic fermentation bycultivating Clostridium botulinum type A in a suitable medium. Raw toxincan be harvested by precipitation with sulfuric acid and concentrated byultramicrofiltration. Purification can be carried out by dissolving theacid precipitate in calcium chloride. The toxin can then be precipitatedwith cold ethanol. The precipitate can be dissolved in sodium phosphatebuffer and centrifuged. Upon drying there can then be obtainedapproximately 900 kD crystalline BoNT/A complex with a specific potencyof 3×10⁷ LD₅₀ U/mg or greater. Furthermore, NAPs can be separated out toobtain purified toxin, such as e.g., BoNT/A with an approximately 150 kDmolecular weight with a specific potency of 1-2×10⁸ LD₅₀ U/mg orgreater, purified BoNT/B with an approximately 156 kD molecular weightwith a specific potency of 1-2×10⁸ LD₅₀ U/mg or greater, and purifiedBoNT/F with an approximately 155 kD molecular weight with a specificpotency of 1-2×10⁷ LD₅₀ U/mg or greater. See Edward J. Schantz & Eric A.Johnson, Properties and use of Botulinum Toxin and Other MicrobialNeurotoxins in Medicine, Microbiol Rev. 56: 80-99 (1992), which ishereby incorporated in its entirety. As another example, recombinantClostridial toxins can be recombinantly produced as described in Stewardet al., Optimizing Expression of Active Botulinum Toxin Type A, U.S.Patent Publication 2008/0057575; and Steward et al., OptimizingExpression of Active Botulinum Toxin Type E, U.S. Patent Publication2008/0138893, each of which is hereby incorporated in its entirety.

Clostridial toxins are also commercially available as pharmaceuticalcompositions include, BoNT/A preparations, such as, e.g., BOTOX®(Allergan, Inc., Irvine, Calif.), DYSPORT®/RELOXIN®, (Beaufour Ipsen,Porton Down, England), NEURONOX® (Medy-Tox, Inc., Ochang-myeon, SouthKorea), BTX-A (Lanzhou Institute Biological Products, China) and XEOMIN®(Merz Pharmaceuticals, GmbH., Frankfurt, Germany); and BoNT/Bpreparations, such as, e.g., MYOBLOC™/NEUROBLOC™ (SolsticeNeurosciences, Inc., South San Francisco, Calif.). Clostridial toxincomplexes may be obtained from, e.g., List Biological Laboratories, Inc.(Campbell, Calif.), the Centre for Applied Microbiology and Research(Porton Down, U.K), Wako (Osaka, Japan), and Sigma Chemicals (St Louis,Mo.).

In an embodiment, a Clostridial may be a Botulinum toxin, Tetanus toxin,a Baratii toxin, or a Butyricum toxin. In aspects of this embodiment, aBotulinum toxin may be a BoNT/A, a BoNT/B, a BoNT/C₁, a BoNT/D, aBoNT/E, a BoNT/F, or a BoNT/G. In another embodiment, a Clostridialtoxin may be a Clostridial toxin variant. In aspects of this embodiment,a Clostridial toxin variant may be a naturally-occurring Clostridialtoxin variant or a non-naturally-occurring Clostridial toxin variant. Inother aspects of this embodiment, a Clostridial toxin variant may be aBoNT/A variant, a BoNT/B variant, a BoNT/C₁ variant, a BoNT/D variant, aBoNT/E variant, a BoNT/F variant, a BoNT/G variant, a TeNT variant, aBaNT variant, or a BuNT variant, where the variant is either anaturally-occurring variant or a non-naturally-occurring variant.

In an embodiment, a Clostridial toxin may be a Clostridial toxincomplex. In aspects of this embodiment, a Clostridial toxin complex maybe a BoNT/A complex, a BoNT/B complex, a BoNT/C₁ complex, a BoNT/Dcomplex, a BoNT/E complex, a BoNT/F complex, a BoNT/G complex, a TeNTcomplex, a BaNT complex, or a BuNT complex. In other aspects of thisembodiment, a Clostridial toxin complex may be a 900-kDa BoNT/A complex,a 500-kDa BoNT/A complex, a 300-kDa BoNT/A complex, a 500-kDa BoNT/Bcomplex, a 500-kDa BoNT/C1 complex, a 500-kDa BoNT/D complex, a 300-kDaBoNT/D complex, a 300-kDa BoNT/E complex, or a 300-kDa BoNT/F complex.

Aspects of the present disclosure comprise, in part, a TargetedExocytosis Modulator. As used herein, the term “Targeted ExocytosisModulator” is synonymous with “TEM” or “retargeted endopeptidase.”Generally, a TEM comprises an enzymatic domain from a Clostridial toxinlight chain, a translocation domain from a Clostridial toxin heavychain, and a targeting domain. The targeting domain of a TEM provides analtered cell targeting capability that targets the molecule to areceptor other than the native Clostridial toxin receptor utilized by anaturally-occurring Clostridial toxin. This re-targeted capability isachieved by replacing the naturally-occurring binding domain of aClostridial toxin with a targeting domain having a binding activity fora non-Clostridial toxin receptor. Although binding to a non-Clostridialtoxin receptor, a TEM undergoes all the other steps of the intoxicationprocess including internalization of the TEM/receptor complex into thecytoplasm, formation of the pore in the vesicle membrane and di-chainmolecule, translocation of the enzymatic domain into the cytoplasm, andexerting a proteolytic effect on a component of the SNARE complex of thetarget cell.

However, an important difference between TEMs, such as, e.g., TEMsdisclosed herein, and native Clostridial toxins is that since TEMs donot target motor neurons, the lethality associated with over-dosing anindividual with a TEM is greatly minimized, if not avoided altogether.For example, a TEM comprising an opioid targeting domain can beadministered at 10,000 times the therapeutically effective dose beforeevidence of lethality is observed, and this lethality is due to thepassive diffusion of the molecule and not via the intoxication process.Thus, for all practical purposes TEMs are non-lethal molecules.

As used herein, the term “Clostridial toxin enzymatic domain” refers toa Clostridial toxin polypeptide located in the light chain of aClostridial toxin that executes the enzymatic target modification stepof the intoxication process. A Clostridial toxin enzymatic domainincludes a metalloprotease region containing a zinc-dependentendopeptidase activity which specifically targets core components of theneurotransmitter release apparatus. Thus, a Clostridial toxin enzymaticdomain specifically targets and proteolytically cleavages of aClostridial toxin substrate, such as, e.g., SNARE proteins like aSNAP-25 substrate, a VAMP substrate and a Syntaxin substrate.

A Clostridial toxin enzymatic domain includes, without limitation,naturally occurring Clostridial toxin enzymatic domain variants, suchas, e.g., Clostridial toxin enzymatic domain isoforms and Clostridialtoxin enzymatic domain subtypes; non-naturally occurring Clostridialtoxin enzymatic domain variants, such as, e.g., conservative Clostridialtoxin enzymatic domain variants, non-conservative Clostridial toxinenzymatic domain variants, Clostridial toxin enzymatic domain chimeras,active Clostridial toxin enzymatic domain fragments thereof, or anycombination thereof. Non-limiting examples of a Clostridial toxinenzymatic domain include, e.g., a BoNT/A enzymatic domain, a BoNT/Benzymatic domain, a BoNT/C1 enzymatic domain, a BoNT/D enzymatic domain,a BoNT/E enzymatic domain, a BoNT/F enzymatic domain, a BoNT/G enzymaticdomain, a TeNT enzymatic domain, a BaNT enzymatic domain, and a BuNTenzymatic domain.

As used herein, the term “Clostridial toxin translocation domain” refersto a Clostridial toxin polypeptide located within the amino-terminalhalf of the heavy chain of a Clostridial toxin that executes thetranslocation step of the intoxication process. The translocation stepappears to involve an allosteric conformational change of thetranslocation domain caused by a decrease in pH within the intracellularvesicle. This conformational change results in the formation of a porein the vesicular membrane that permits the movement of the light chainfrom within the vesicle into the cytoplasm. Thus, a Clostridial toxintranslocation domain facilitates the movement of a Clostridial toxinlight chain across a membrane of an intracellular vesicle into thecytoplasm of a cell.

A Clostridial toxin translocation domain includes, without limitation,naturally occurring Clostridial toxin translocation domain variants,such as, e.g., Clostridial toxin translocation domain isoforms andClostridial toxin translocation domain subtypes; non-naturally occurringClostridial toxin translocation domain variants, such as, e.g.,conservative Clostridial toxin translocation domain variants,non-conservative Clostridial toxin translocation domain variants,Clostridial toxin translocation domain chimerics, active Clostridialtoxin translocation domain fragments thereof, or any combinationthereof. Non-limiting examples of a Clostridial toxin translocationdomain include, e.g., a BoNT/A translocation domain, a BoNT/Btranslocation domain, a BoNT/C1 translocation domain, a BoNT/Dtranslocation domain, a BoNT/E translocation domain, a BoNT/Ftranslocation domain, a BoNT/G translocation domain, a TeNTtranslocation domain, a BaNT translocation domain, and a BuNTtranslocation domain.

As used herein, the term “targeting domain” is synonymous with “bindingdomain” or “targeting moiety” and refers to a polypeptide that executesthe receptor binding and/or complex internalization steps of theintoxication process, with the proviso that the binding domain is not aClostridial toxin binding domain found within the carboxyl-terminal halfof the heavy chain of a Clostridial toxin. A targeting domain includes areceptor binding region that confers the binding activity and/orspecificity of the targeting domain for its cognate receptor. As usedherein, the term “cognate receptor” refers to a receptor for which thetargeting domain preferentially interacts with under physiologicalconditions, or under in vitro conditions substantially approximatingphysiological conditions. As used herein, the term “preferentiallyinteracts” is synonymous with “preferentially binding” and refers to aninteraction that is statistically significantly greater in degreerelative to a control. With reference to a targeting domain disclosedherein, a targeting domain binds to its cognate receptor to astatistically significantly greater degree relative to a non-cognatereceptor. Said another way, there is a discriminatory binding of thetargeting domain to its cognate receptor relative to a non- cognatereceptor. Thus, a targeting domain directs binding to a TEM-specificreceptor located on the plasma membrane surface of a target cell.

In an embodiment, a targeting domain disclosed herein has an associationrate constant that confers preferential binding to its cognate receptor.In aspects of this embodiment, a targeting domain disclosed herein bindsto its cognate receptor with an association rate constant of, e.g., lessthan 1×10⁵ M⁻¹ s⁻¹, less than 1×10⁶ M⁻¹ s⁻¹, less than 1×10⁷ M⁻¹ s⁻¹, orless than 1×10⁸ M⁻¹ s⁻¹. In other aspects of this embodiment, atargeting domain disclosed herein binds to its cognate receptor with anassociation rate constant of, e.g., more than 1×10⁵ M⁻¹ s⁻¹, more than1×10⁶ M⁻¹ s⁻¹, more than 1×10⁷ M⁻¹ s⁻¹, or more than 1×10⁸ M⁻¹ s⁻¹. Inyet other aspects of this embodiment, a targeting domain disclosedherein binds to its cognate receptor with an association rate constantbetween 1×10⁵ M⁻¹ s⁻¹ to 1×10⁸ M⁻¹ s⁻¹, 1×10⁶ M⁻¹ s⁻¹ to 1×10⁸ M⁻¹ s⁻¹,1×10⁵ M⁻¹ s⁻¹ to 1×10⁷ M⁻¹ s⁻¹, or 1×10⁶ M⁻¹ s⁻¹ to 1×10⁷ M⁻¹ s⁻¹.

In another embodiment, a targeting domain disclosed herein has anassociation rate constant that is greater for its cognate targetreceptor relative to a non-cognate receptor. In other aspects of thisembodiment, a targeting domain disclosed herein has an association rateconstant that is greater for its cognate target receptor relative to anon-cognate receptor by, at least one-fold, at least two-fold, at leastthree-fold, at least four fold, at least five-fold, at least 10 fold, atleast 50 fold, at least 100 fold, at least 1000 fold, at least 10,000fold, or at least 100,000 fold. In other aspects of this embodiment, atargeting domain disclosed herein has an association rate constant thatis greater for its cognate target receptor relative to a non-cognatereceptor by, e.g., about one-fold to about three-fold, about one-fold toabout five-fold, about one-fold to about 10-fold, about one-fold toabout 100-fold, about one-fold to about 1000-fold, about five-fold toabout 10-fold, about five-fold to about 100-fold, about five-fold toabout 1000-fold, about 10-fold to about 100-fold, about 10-fold to about1000-fold, about 10-fold to about 10,000-fold, or about 10-fold to about100,000-fold.

In yet another embodiment, a targeting domain disclosed herein has adisassociation rate constant that confers preferential binding to itscognate receptor. In other aspects of this embodiment, a targetingdomain disclosed herein binds to its cognate receptor with adisassociation rate constant of less than 1×10⁻³ s⁻¹, less than 1×10⁻⁴s⁻¹, or less than 1×10⁻⁵ s⁻¹. In yet other aspects of this embodiment, atargeting domain disclosed herein binds to its cognate receptor with adisassociation rate constant of, e.g., less than 1.0×10⁻⁴ s⁻¹, less than2.0×10⁻⁴ s⁻¹, less than 3.0×10⁻⁴ s⁻¹, less than 4.0×10⁻⁴ s⁻¹, less than5.0×10⁻⁴ s⁻¹, less than 6.0×10⁻⁴ s⁻¹, less than 7.0×10⁻⁴ s⁻¹, less than8.0×10⁻⁴ s⁻¹, or less than 9.0×10⁻⁴ s⁻¹. In still other aspects of thisembodiment, a targeting domain disclosed herein binds to its cognatereceptor with a disassociation rate constant of, e.g., more than 1×10⁻³s⁻¹, more than 1×10⁻⁴ s⁻¹, or more than 1×10⁻⁵ s⁻¹. In other aspects ofthis embodiment, a targeting domain disclosed herein binds to itscognate receptor with a disassociation rate constant of, e.g., more than1.0×10⁻⁴ s⁻¹, more than 2.0×10⁻⁴ s⁻¹, more than 3.0×10⁻⁴ s⁻¹, more than4.0×10⁻⁴ s⁻¹, more than 5.0×10⁻⁴ s⁻¹, more than 6.0×10⁻⁴ s⁻¹, more than7.0×10⁻⁴ s⁻¹, more than 8.0×10 ⁻⁴ s⁻¹, or more than 9.0×10⁻⁴ s⁻¹.

In still another embodiment, a targeting domain disclosed herein has adisassociation rate constant that is less for its cognate targetreceptor relative to a non-cognate receptor. In other aspects of thisembodiment, a targeting domain disclosed herein has a disassociationrate constant that is less for its cognate target receptor relative to anon-cognate receptor by, e.g., at least one-fold, at least two-fold, atleast three-fold, at least four fold, at least five-fold, at least 10fold, at least 50 fold, at least 100 fold, at least 1000 fold, at least10,000 fold, or at least 100,000 fold. In other aspects of thisembodiment, a targeting domain disclosed herein has a disassociationrate constant that is less for its cognate target receptor relative to anon-cognate receptor by, e.g., about one-fold to about three-fold, aboutone-fold to about five-fold, about one-fold to about 10-fold, aboutone-fold to about 100-fold, about one-fold to about 1000-fold, aboutfive-fold to about 10-fold, about five-fold to about 100-fold, aboutfive-fold to about 1000-fold, about 10-fold to about 100-fold, about10-fold to about 1000-fold, about 10-fold to about 10,000-fold, or about10-fold to about 100,000-fold.

In another embodiment, a targeting domain disclosed herein has anequilibrium disassociation constant that confers preferential binding toits cognate receptor. In other aspects of this embodiment, a targetingdomain disclosed herein binds to its cognate receptor with anequilibrium disassociation constant of, e.g., less than 0.500 nM. In yetother aspects of this embodiment, a targeting domain disclosed hereinbinds to its cognate receptor with an equilibrium disassociationconstant of, e.g., less than 0.500 nM, less than 0.450 nM, less than0.400 nM, less than 0.350 nM, less than 0.300 nM, less than 0.250 nM,less than 0.200 nM, less than 0.150 nM, less than 0.100 nM, or less than0.050 nM. In other aspects of this embodiment, a targeting domaindisclosed herein binds to its cognate receptor with an equilibriumdisassociation constant of, e.g., more than 0.500 nM, more than 0.450nM, more than 0.400 nM, more than 0.350 nM, more than 0.300 nM, morethan 0.250 nM, more than 0.200 nM, more than 0.150 nM, more than 0.100nM, or more than 0.050 nM.

In yet another embodiment, a targeting domain disclosed herein has anequilibrium disassociation constant that is greater for its cognatetarget receptor relative to a non-cognate receptor. In other aspects ofthis embodiment, a targeting domain disclosed herein has an equilibriumdisassociation constant that is greater for its cognate target receptorrelative to a non-cognate receptor by, e.g., at least one-fold, at leasttwo-fold, at least three-fold, at least four fold, at least five-fold,at least 10 fold, at least 50 fold, at least 100 fold, at least 1000fold, at least 10,000 fold, or at least 100,000 fold. In other aspectsof this embodiment, a targeting domain disclosed herein has anequilibrium disassociation constant that is greater for its cognatetarget receptor relative to a non-cognate receptor by, e.g., aboutone-fold to about three-fold, about one-fold to about five-fold, aboutone-fold to about 10-fold, about one-fold to about 100-fold, aboutone-fold to about 1000-fold, about five-fold to about 10-fold, aboutfive-fold to about 100-fold, about five-fold to about 1000-fold, about10-fold to about 100-fold, about 10-fold to about 1000-fold, about10-fold to about 10,000-fold, or about 10-fold to about 100,000-fold.

In another embodiment, a targeting domain disclosed herein may be onethat preferentially interacts with a receptor located on a sensoryneuron. In an aspect of this embodiment, the sensory neuron targetingdomain is one whose cognate receptor is located exclusively on theplasma membrane of sensory neurons. In another aspect of thisembodiment, the sensory neuron targeting domain is one whose cognatereceptor is located primarily on the plasma membrane of sensory neuron.For example, a receptor for a sensory neuron targeting domain is locatedprimarily on a sensory neuron when, e.g., at least 60% of all cells thathave a cognate receptor for a sensory neuron targeting domain on thesurface of the plasma membrane are sensory neurons, at least 70% of allcells that have a cognate receptor for a sensory neuron targeting domainon the surface of the plasma membrane are sensory neurons, at least 80%of all cells that have a cognate receptor for a sensory neuron targetingdomain on the surface of the plasma membrane are sensory neurons, or atleast 90% of all cells that have a cognate receptor for a sensory neurontargeting domain on the surface of the plasma membrane are sensoryneurons. In yet another aspect of this embodiment, the sensory neurontargeting domain is one whose cognate receptor is located on the plasmamembrane of several types of cells, including sensory neurons. In stillanother aspect of this embodiment, the sensory neuron targeting domainis one whose cognate receptor is located on the plasma membrane ofseveral types of cells, including sensory neurons, with the proviso thatmotor neurons are not one of the other types of cells.

In another embodiment, a targeting domain disclosed herein may be onethat preferentially interacts with a receptor located on a sympatheticneuron. In an aspect of this embodiment, the sympathetic neurontargeting domain is one whose cognate receptor is located exclusively onthe plasma membrane of sympathetic neurons. In another aspect of thisembodiment, the sympathetic neuron targeting domain is one whose cognatereceptor is located primarily on the plasma membrane of sympatheticneuron. For example, a receptor for a sympathetic neuron targetingdomain is located primarily on a sympathetic neuron when, e.g., at least60% of all cells that have a cognate receptor for a sympathetic neurontargeting domain on the surface of the plasma membrane are sympatheticneurons, at least 70% of all cells that have a cognate receptor for asympathetic neuron targeting domain on the surface of the plasmamembrane are sympathetic neurons, at least 80% of all cells that have acognate receptor for a sympathetic neuron targeting domain on thesurface of the plasma membrane are sympathetic neurons, or at least 90%of all cells that have a cognate receptor for a sympathetic neurontargeting domain on the surface of the plasma membrane are sympatheticneurons. In yet another aspect of this embodiment, the sympatheticneuron targeting domain is one whose cognate receptor is located on theplasma membrane of several types of cells, including sympatheticneurons. In still another aspect of this embodiment, the sympatheticneuron targeting domain is one whose cognate receptor is located on theplasma membrane of several types of cells, including sympatheticneurons, with the proviso that motor neurons are not one of the othertypes of cells.

In another embodiment, a targeting domain disclosed herein may be onethat preferentially interacts with a receptor located on aparasympathetic neuron. In an aspect of this embodiment, theparasympathetic neuron targeting domain is one whose cognate receptor islocated exclusively on the plasma membrane of parasympathetic neurons.In another aspect of this embodiment, the parasympathetic neurontargeting domain is one whose cognate receptor is located primarily onthe plasma membrane of parasympathetic neuron. For example, a receptorfor a parasympathetic neuron targeting domain is located primarily on aparasympathetic neuron when, e.g., at least 60% of all cells that have acognate receptor for a parasympathetic neuron targeting domain on thesurface of the plasma membrane are parasympathetic neurons, at least 70%of all cells that have a cognate receptor for a parasympathetic neurontargeting domain on the surface of the plasma membrane areparasympathetic neurons, at least 80% of all cells that have a cognatereceptor for a parasympathetic neuron targeting domain on the surface ofthe plasma membrane are parasympathetic neurons, or at least 90% of allcells that have a cognate receptor for a parasympathetic neurontargeting domain on the surface of the plasma membrane areparasympathetic neurons. In yet another aspect of this embodiment, theparasympathetic neuron targeting domain is one whose cognate receptor islocated on the plasma membrane of several types of cells, includingparasympathetic neurons. In still another aspect of this embodiment, theparasympathetic neuron targeting domain is one whose cognate receptor islocated on the plasma membrane of several types of cells, includingparasympathetic neurons, with the proviso that motor neurons are not oneof the other types of cells.

In another embodiment, a targeting domain disclosed herein is an opioidpeptide targeting domain, a galanin peptide targeting domain, a PARpeptide targeting domain, a somatostatin peptide targeting domain, aneurotensin peptide targeting domain, a SLURP peptide targeting domain,an angiotensin peptide targeting domain, a tachykinin peptide targetingdomain, a Neuropeptide Y related peptide targeting domain, a kininpeptide targeting domain, a melanocortin peptide targeting domain, or agranin peptide targeting domain, a glucagon like hormone peptidetargeting domain, a secretin peptide targeting domain, a pituitaryadenylate cyclase activating peptide (PACAP) peptide targeting domain, agrowth hormone-releasing hormone (GHRH) peptide targeting domain, avasoactive intestinal peptide (VIP) peptide targeting domain, a gastricinhibitory peptide (GIP) peptide targeting domain, a calcitonin peptidetargeting domain, a visceral gut peptide targeting domain, aneurotrophin peptide targeting domain, a head activator (HA) peptide, aglial cell line-derived neurotrophic factor (GDNF) family of ligands(GFL) peptide targeting domain, a RF-amide related peptide (RFRP)peptide targeting domain, a neurohormone peptide targeting domain, or aneuroregulatory cytokine peptide targeting domain, an interleukin (IL)targeting domain, vascular endothelial growth factor (VEGF) targetingdomain, an insulin-like growth factor (IGF) targeting domain, anepidermal growth factor (EGF) targeting domain, a Transformation GrowthFactor-β (TGF-β) targeting domain, a Bone Morphogenetic Protein (BMP)targeting domain, a Growth and Differentiation Factor (GDF) targetingdomain, an activin targeting domain, or a Fibroblast Growth Factor (FGF)targeting domain, or a Platelet-Derived Growth Factor (PDGF) targetingdomain.

In an aspect of this embodiment, an opioid peptide targeting domain isan enkephalin peptide, a bovine adrenomedullary-22 (BAM22) peptide, anendomorphin peptide, an endorphin peptide, a dynorphin peptide, anociceptin peptide, or a hemorphin peptide. In another aspect of thisembodiment, an enkephalin peptide targeting domain is a Leu-enkephalinpeptide, a Met-enkephalin peptide, a Met-enkephalin MRGL peptide, or aMet-enkephalin MRF peptide. In another aspect of this embodiment, abovine adrenomedullary-22 peptide targeting domain is a BAM22 (1-12)peptide, a BAM22 (6-22) peptide, a BAM22 (8-22) peptide, or a BAM22(1-22) peptide. In another aspect of this embodiment, an endomorphinpeptide targeting domain is an endomorphin-1 peptide or an endomorphin-2peptide. In another aspect of this embodiment, an endorphin peptidetargeting domain an endorphin-α peptide, a neoendorphin-α peptide, anendorphin-β peptide, a neoendorphin-β peptide, or an endorphin-γpeptide. In another aspect of this embodiment, a dynorphin peptidetargeting domain is a dynorphin A peptide, a dynorphin B (leumorphin)peptide, or a rimorphin peptide. In another aspect of this embodiment, anociceptin peptide targeting domain is a nociceptin RK peptide, anociceptin peptide, a neuropeptide 1 peptide, a neuropeptide 2 peptide,or a neuropeptide 3 peptide. In another aspect of this embodiment, ahemorphin peptide targeting domain is a LVVH7 peptide, a VVH7 peptide, aVH7 peptide, a H7 peptide, a LVVH6 peptide, a LVVH5 peptide, a VVH5peptide, a LVVH4 peptide, or a LVVH3 peptide.

In an aspect of this embodiment, a galanin peptide targeting domain is agalanin peptide, a galanin message-associated peptide (GMAP) peptide, agalanin like protein (GALP) peptide, or an alarin peptide.

In an aspect of this embodiment, a PAR peptide targeting domain is aPAR1 peptide, a PAR2 peptide, a PAR3 peptide and a PAR4 peptide. In anaspect of this embodiment, a somatostatin peptide targeting domain is asomatostatin peptide or a cortistatin peptide. In an aspect of thisembodiment, a neurotensin peptide targeting domain a neurotensin or aneuromedin N. In an aspect of this embodiment, a SLURP peptide targetingdomain is a SLURP-1 peptide or a SLURP-2 peptide. In an aspect of thisembodiment, an angiotensin peptide targeting domain is an angiotensinpeptide.

In an aspect of this embodiment, a tachykinin peptide targeting domainis a Substance P peptide, a neuropeptide K peptide, a neuropeptide gammapeptide, a neurokinin A peptide, a neurokinin B peptide, a hemokininpeptide, or a endokinin peptide. In an aspect of this embodiment, aNeuropeptide Y related peptide targeting domain is a Neuropeptide Ypeptide, a Peptide YY peptide, Pancreatic peptide, a Pancreaticicosapeptide peptide, a Pancreatic Hormone domain peptide, a CXCL12peptide, and a Sjogren syndrome antigen B peptide. In an aspect of thisembodiment, a kinin peptide targeting domain is a bradykinin peptide, akallidin peptide, a desArg9 bradykinin peptide, a desArg10 bradykininpeptide, a kininogen peptide, gonadotropin releasing hormone 1 peptide,chemokine peptide, an arginine vasopressin peptide.

In an aspect of this embodiment, a melanocortin peptide targeting domaincomprises a melanocyte stimulating hormone peptide, anadrenocorticotropin peptide, a lipotropin peptide, or a melanocortinpeptide derived neuropeptide. In an aspect of this embodiment, amelanocyte stimulating hormone peptide targeting domain comprises ana-melanocyte stimulating hormone peptide, a β-melanocyte stimulatinghormone peptide, or a γ-melanocyte stimulating hormone peptide. In anaspect of this embodiment, an adrenocorticotropin peptide targetingdomain comprises an adrenocorticotropin or a Corticotropin-likeintermediary peptide. In an aspect of this embodiment, a lipotropinpeptide targeting domain comprises a β-lipotropin peptide or aγ-lipotropin peptide.

In an aspect of this embodiment, a granin peptide targeting domaincomprises a chromogranin A peptide, a chromogranin B peptide, achromogranin C (secretogranin II) peptide, a secretogranin IV peptide,or a secretogranin VI peptide. In an aspect of this embodiment, achromogranin A peptide targeting domain comprises a β-granin peptide, avasostatin peptide, a chromostatin peptide, a pancreastatin peptide, aWE-14 peptide, a catestatin peptide, a parastatin peptide, or a GE-25peptide. In an aspect of this embodiment, a chromogranin B peptidetargeting domain comprises a GAWK peptide, an adrenomedullary peptide,or a secretolytin peptide. In an aspect of this embodiment, achromogranin C peptide targeting domain comprises a secretoneurinpeptide.

In an aspect of this embodiment, a glucagons-like hormone peptidetargeting domain is a glucagon-like peptide-1, a glucagon-likepeptide-2, a glicentin, a glicentin-related peptide (GRPP), a glucagon,or an oxyntomodulin (OXY). In an aspect of this embodiment, a secretinpeptide targeting domain is a secretin peptide. In an aspect of thisembodiment, a pituitary adenylate cyclase activating peptide targetingdomain is a pituitary adenylate cyclase activating peptide. In an aspectof this embodiment, a growth hormone-releasing hormone peptide targetingdomain a growth hormone-releasing hormone peptide. In an aspect of thisembodiment, a vasoactive intestinal peptide targeting domain is avasoactive intestinal peptide-1 peptide or a vasoactive intestinalpeptide-2 peptide. In an aspect of this embodiment, a gastric inhibitorypeptide targeting domain is a gastric inhibitory peptide. In an aspectof this embodiment, a calcitonin peptide targeting domain is acalcitonin peptide, an amylin peptide, a calcitonin-related peptide a, acalcitonin-related peptide β, and a islet amyloid peptide. In an aspectof this embodiment, a visceral gut peptide targeting domain is a gastrinpeptide, a gastrin-releasing peptide, or a cholecystokinin peptide.

In an aspect of this embodiment, a neurotrophin peptide targeting domainis a nerve growth factor (NGF) peptide, a brain derived neurotrophicfactor (BDNF) peptide, a neurotrophin-3 (NT-3) peptide, aneurotrophin-4/5 (NT-4/5) peptide, or an amyloid beta (A4) precursorprotein neurotrophin (APP) peptide. In an aspect of this embodiment, ahead activator peptide targeting domain is a head activator peptide. Inan aspect of this embodiment, a glial cell line-derived neurotrophicfactor family of ligands peptide targeting domain is a glial cellline-derived neurotrophic factor peptide, a Neurturin peptide, aPersephin peptide, or an Artemin peptide. In an aspect of thisembodiment, a RF-amide related peptide targeting domain a RF-amiderelated peptide-1, a RF-amide related peptide-2, a RF-amide relatedpeptide-3, a neuropeptide AF, or a neuropeptide FF.

In an aspect of this embodiment, a neurohormone peptide targeting domainis a corticotropin-releasing hormone (CCRH), a parathyroid hormone(PTH), a parathyroid hormone-like hormone (PTHLH), a PHYH, athyrotropin-releasing hormone (TRH), an urocortin-1 (UCN1), anurocortin-2 (UCN2), an urocortin-3 (UCN3), or an urotensin-2 (UTS2). Inan aspect of this embodiment, a neuroregulatory cytokine peptidetargeting domain is a ciliary neurotrophic factor peptide, aglycophorin-A peptide, a leukemia inhibitory factor peptide, acardiotrophin-1 peptide, a cardiotrophin-like cytokine peptide, aneuroleukin peptide, and an oncostatin M peptide. In an aspect of thisembodiment, an IL peptide targeting domain is an IL-1 peptide, an IL-2peptide, an IL-3 peptide, an IL-4 peptide, an IL-5 peptide, an IL-6peptide, an IL-7 peptide, an IL-8 peptide, an IL-9 peptide, an IL-10peptide, an IL-11 peptide, an IL-12 peptide, an IL-18 peptide, an IL-32peptide, or an IL-33 peptide.

In an aspect of this embodiment, a VEGF peptide targeting domain is aVEGF-A peptide, a VEGF-B peptide, a VEGF-C peptide, a VEGF-D peptide, ora placenta growth factor (PIGF) peptide. In an aspect of thisembodiment, an IGF peptide targeting domain is an IGF-1 peptide or anIGF-2 peptide. In an aspect of this embodiment, an EGF peptide targetingdomain an EGF, a heparin-binding EGF-like growth factor (HB-EGF), atransforming growth factor-α (TGF-α), an amphiregulin (AR), anepiregulin (EPR), an epigen (EPG), a betacellulin (BTC), a neuregulin-1(NRG1), a neuregulin-2 (NRG2), a neuregulin-3, (NRG3), or a neuregulin-4(NRG4). In an aspect of this embodiment, a FGF peptide targeting domainis a FGF1 peptide, a FGF2 peptide, a FGF3 peptide, a FGF4 peptide, aFGF5 peptide, a FGF6 peptide, a FGF7 peptide, a FGF8 peptide, a FGF9peptide, a FGF10 peptide, a FGF17 peptide, or a FGF18 peptide. In anaspect of this embodiment, a PDGF peptide targeting domain is a PDGFαpeptide or a PDGFβ peptide.

In an aspect of this embodiment, a TGFβ peptide targeting domain is aTGFβ1 peptide, a TGFβ2 peptide, a TGFβ3 peptide, or a TGFβ4 peptide. Inan aspect of this embodiment, a BMP peptide targeting domain is a BMP2peptide, a BMP3 peptide, a BMP4 peptide, a BMP5 peptide, a BMP6 peptide,a BMP7 peptide, a BMP8 peptide, or a BMP10 peptide. In an aspect of thisembodiment, a GDF peptide targeting domain is a GDF1 peptide, a GDF2peptide, a GDF3 peptide, a GDF5 peptide, a GDF6 peptide, a GDF7 peptide,a GDF8 peptide, a GDF10 peptide, a GDF11 peptide, or a GDF15 peptide. Inan aspect of this embodiment, an activin peptide targeting domain is anactivin A peptide, an activin B peptide, an activin C peptide, anactivin E peptide, or an inhibin A peptide.

As discussed above, naturally-occurring Clostridial toxins are organizedinto three functional domains comprising a linear amino-to-carboxylsingle polypeptide order of the enzymatic domain (amino regionposition), the translocation domain (middle region position) and thebinding domain (carboxyl region position) (FIG. 2). Thisnaturally-occurring order can be referred to as the carboxylpresentation of the binding domain because the domain necessary forbinding to the receptor is located at the carboxyl region position ofthe Clostridial toxin. However, it has been shown that Clostridialtoxins can be modified by rearranging the linear amino-to-carboxylsingle polypeptide order of the three major domains and locating atargeting moiety at the amino region position of a Clostridial toxin,referred to as amino presentation, as well as in the middle regionposition, referred to as central presentation (FIG. 4).

Thus, a TEM can comprise a targeting domain in any and all locationswith the proviso that TEM is capable of performing the intoxicationprocess. Non-limiting examples include, locating a targeting domain atthe amino terminus of a TEM; locating a targeting domain between aClostridial toxin enzymatic domain and a Clostridial toxin translocationdomain of a TEM; and locating a targeting domain at the carboxylterminus of a TEM. Other non-limiting examples include, locating atargeting domain between a Clostridial toxin enzymatic domain and aClostridial toxin translocation domain of a TEM. The enzymatic domain ofnaturally-occurring Clostridial toxins contains the native startmethionine. Thus, in domain organizations where the enzymatic domain isnot in the amino-terminal location an amino acid sequence comprising thestart methionine should be placed in front of the amino-terminal domain.Likewise, where a targeting domain is in the amino-terminal position, anamino acid sequence comprising a start methionine and a proteasecleavage site may be operably-linked in situations in which a targetingdomain requires a free amino terminus, see, e.g., Shengwen Li et al.,Degradable Clostridial Toxins, U.S. patent application Ser. No.11/572,512 (Jan. 23, 2007), which is hereby incorporated by reference inits entirety. In addition, it is known in the art that when adding apolypeptide that is operably-linked to the amino terminus of anotherpolypeptide comprising the start methionine that the original methionineresidue can be deleted.

A TEM disclosed herein may optionally comprise an exogenous proteasecleavage site that allows the use of an exogenous protease to convertthe single-chain polypeptide form of a TEM into its more active di-chainform. As used herein, the term “exogenous protease cleavage site” issynonymous with a “non-naturally occurring protease cleavage site” or“non-native protease cleavage site” and means a protease cleavage sitethat is not naturally found in a di-chain loop region from a naturallyoccurring Clostridial toxin.

Naturally-occurring Clostridial toxins are each translated as asingle-chain polypeptide of approximately 150 kDa that is subsequentlycleaved by proteolytic scission within a disulfide loop by anaturally-occurring protease (FIG. 2). This cleavage occurs within thediscrete di-chain loop region located between two cysteine residues thatform a disulfide bridge and comprising an endogenous protease cleavagesite. As used herein, the term “endogenous di-chain loop proteasecleavage site” is synonymous with a “naturally occurring di-chain loopprotease cleavage site” and refers to a naturally occurring proteasecleavage site found within the di-chain loop region of a naturallyoccurring Clostridial toxin. This post-translational processing yields adi-chain molecule comprising an approximately 50 kDa light chain,comprising the enzymatic domain, and an approximately 100 kDa heavychain, comprising the translocation and cell binding domains, the lightchain and heavy chain being held together by the single disulfide bondand non-covalent interactions (FIG. 2). Recombinantly-producedClostridial toxins generally substitute the naturally-occurring di-chainloop protease cleavage site with an exogenous protease cleavage site tofacilitate production of a recombinant di-chain molecule (FIGS. 3-5).See e.g., Dolly, J. O. et al., Activatable Clostridial Toxins, U.S. Pat.No. 7,419,676 (Sep. 2, 2008), which is hereby incorporated by reference.

Although TEMs vary in their overall molecular weight because the size ofthe targeting domain, the activation process and its reliance on anexogenous cleavage site is essentially the same as that forrecombinantly-produced Clostridial toxins. See e.g., Steward, et al.,Activatable Clostridial Toxins, US 2009/0081730; Steward, et al.,Modified Clostridial Toxins with Enhanced Translocation Capabilities andAltered Targeting Activity For Non-Clostridial Toxin Target Cells, U.S.patent application Ser. No. 11/776,075; Steward, et al., ModifiedClostridial Toxins with Enhanced Translocation Capabilities and AlteredTargeting Activity for Clostridial Toxin Target Cells, US 2008/0241881,each of which is hereby incorporated by reference. In general, theactivation process that converts the single-chain polypeptide into itsdi-chain form using exogenous proteases can be used to process TEMshaving a targeting domain organized in an amino presentation, centralpresentation, or carboxyl presentation arrangement. This is because formost targeting domains the amino-terminus of the moiety does notparticipate in receptor binding. As such, a wide range of proteasecleavage sites can be used to produce an active di-chain form of a TEM.However, targeting domains requiring a free amino-terminus for receptorbinding require a protease cleavage site whose scissile bond is locatedat the carboxyl terminus. The use of protease cleavage site is thedesign of a TEM are described in, e.g., Steward, et al., ActivatableClostridial toxins, US 2009/0069238; Ghanshani, et al., ModifiedClostridial Toxins Comprising an Integrated Protease CleavageSite-Binding Domain, US 2011/0189162; and Ghanshani, et al., Methods ofIntracellular Conversion of Single-Chain Proteins into their Di-chainForm, International Patent Application Serial No. PCT/US2011/22272, eachof which is incorporated by reference in its entirety.

Non-limiting examples of exogenous protease cleavage sites include,e.g., a plant papain cleavage site, an insect papain cleavage site, acrustacean papain cleavage site, an enterokinase protease cleavage site,a Tobacco Etch Virus protease cleavage site, a Tobacco Vein MottlingVirus protease cleavage site, a human rhinovirus 3C protease cleavagesite, a human enterovirus 3C protease cleavage site, a subtilisincleavage site, a hydroxylamine cleavage site, a SUMO/ULP-1 proteasecleavage site, and a Caspase 3 cleavage site.

Thus, in an embodiment, a TEM can comprise an amino to carboxyl singlepolypeptide linear order comprising a targeting domain, a translocationdomain, an exogenous protease cleavage site and an enzymatic domain(FIG. 3A). In an aspect of this embodiment, a TEM can comprise an aminoto carboxyl single polypeptide linear order comprising a targetingdomain, a Clostridial toxin translocation domain, an exogenous proteasecleavage site and a Clostridial toxin enzymatic domain.

In another embodiment, a TEM can comprise an amino to carboxyl singlepolypeptide linear order comprising a targeting domain, an enzymaticdomain, an exogenous protease cleavage site, and a translocation domain(FIG. 3B). In an aspect of this embodiment, a TEM can comprise an aminoto carboxyl single polypeptide linear order comprising a targetingdomain, a Clostridial toxin enzymatic domain, an exogenous proteasecleavage site, a Clostridial toxin translocation domain.

In yet another embodiment, a TEM can comprise an amino to carboxylsingle polypeptide linear order comprising an enzymatic domain, anexogenous protease cleavage site, a targeting domain, and atranslocation domain (FIG. 4A). In an aspect of this embodiment, a TEMcan comprise an amino to carboxyl single polypeptide linear ordercomprising a Clostridial toxin enzymatic domain, an exogenous proteasecleavage site, a targeting domain, and a Clostridial toxin translocationdomain.

In yet another embodiment, a TEM can comprise an amino to carboxylsingle polypeptide linear order comprising a translocation domain, anexogenous protease cleavage site, a targeting domain, and an enzymaticdomain (FIG. 4B). In an aspect of this embodiment, a TEM can comprise anamino to carboxyl single polypeptide linear order comprising aClostridial toxin translocation domain, a targeting domain, an exogenousprotease cleavage site and a Clostridial toxin enzymatic domain.

In another embodiment, a TEM can comprise an amino to carboxyl singlepolypeptide linear order comprising an enzymatic domain, a targetingdomain, an exogenous protease cleavage site, and a translocation domain(FIG. 4C). In an aspect of this embodiment, a TEM can comprise an aminoto carboxyl single polypeptide linear order comprising a Clostridialtoxin enzymatic domain, a targeting domain, an exogenous proteasecleavage site, a Clostridial toxin translocation domain.

In yet another embodiment, a TEM can comprise an amino to carboxylsingle polypeptide linear order comprising a translocation domain, atargeting domain, an exogenous protease cleavage site and an enzymaticdomain (FIG. 4D). In an aspect of this embodiment, a TEM can comprise anamino to carboxyl single polypeptide linear order comprising aClostridial toxin translocation domain, a targeting domain, an exogenousprotease cleavage site and a Clostridial toxin enzymatic domain.

In still another embodiment, a TEM can comprise an amino to carboxylsingle polypeptide linear order comprising an enzymatic domain, anexogenous protease cleavage site, a translocation domain, and atargeting domain (FIG. 5A). In an aspect of this embodiment, a TEM cancomprise an amino to carboxyl single polypeptide linear order comprisinga Clostridial toxin enzymatic domain, an exogenous protease cleavagesite, a Clostridial toxin translocation domain, and a targeting domain.

In still another embodiment, a TEM can comprise an amino to carboxylsingle polypeptide linear order comprising a translocation domain, anexogenous protease cleavage site, an enzymatic domain and a targetingdomain, (FIG. 5B). In an aspect of this embodiment, a TEM can comprisean amino to carboxyl single polypeptide linear order comprising aClostridial toxin translocation domain, a targeting domain, an exogenousprotease cleavage site and a Clostridial toxin enzymatic domain.

Non-limiting examples of TEMs disclosed herein, including TEMscomprising a Clostridial toxin enzymatic domain, a Clostridial toxintranslocation domain and a targeting domain, the use of an exogenousprotease cleavage site, and the design of amino presentation, centralpresentation and carboxyl presentation TEMs are described in, e.g., U.S.Pat. No. 7,959,933, Activatable Recombinant Neurotoxins, U.S. Pat. No.7,897,157, Activatable Clostridial Toxins; U.S. Pat. No. 7,833,535,Clostridial Toxin Derivatives and Methods for Treating Pain; U.S. Pat.No. 7,811,584, Multivalent Clostridial Toxins; U.S. Pat. No. 7,780,968,Clostridial Toxin Derivatives and Methods for Treating Pain; U.S. Pat.No. 7,749,514, Activatable Clostridial Toxins, U.S. Pat. No. 7,740,868,Activatable Clostridial Toxins; U.S. Pat. No. 7,736,659, ClostridialToxin Derivatives and Methods for Treating Pain; U.S. Pat. No.7,709,228, Activatable Recombinant Neurotoxins; U.S. Pat. No. 7,704,512,Clostridial Toxin Derivatives and Methods for Treating Pain; U.S. Pat.No. 7,659,092, Fusion Proteins; U.S. Pat. No. 7,658,933, Non-CytotoxicProtein Conjugates; U.S. Pat. No. 7,622,127, Clostridial ToxinDerivatives and Methods for Treating Pain; U.S. Pat. No. 7,514,088,Multivalent Clostridial Toxin Derivatives and Methods of Their Use; U.S.Pat. No. 7,425,338, Clostridial Toxin Derivatives and Methods forTreating Pain; U.S. Pat. No. 7,422,877, Activatable RecombinantNeurotoxins; U.S. Pat. No. 7,419,676, Activatable RecombinantNeurotoxins; U.S. Pat. No. 7,413,742, Clostridial Toxin Derivatives andMethods for Treating Pain; U.S. Pat. No. 7,262,291, Clostridial ToxinDerivatives and Methods for Treating Pain; U.S. Pat. No. 7,244,437,Clostridial Toxin Derivatives and Methods for Treating Pain; U.S. Pat.No. 7,244,436, Clostridial Toxin Derivatives and Methods for TreatingPain; U.S. Pat. No. 7,138,127, Clostridial Toxin Derivatives and Methodsfor Treating Pain; U.S. Pat. No. 7,132,259, Activatable RecombinantNeurotoxins; U.S. Pat. No. 7,056,729, Botulinum Neurotoxin-Substance PConjugate or Fusion Protein for Treating Pain; U.S. Pat. No. 6,641,820,Clostridial Toxin Derivatives and Methods to Treat Pain; U.S. Pat. No.6,500,436, Clostridial Toxin Derivatives and Methods for Treating Pain;US 2011/0091437, Fusion Proteins; US 2011/0070621, MultivalentClostridial Toxins; US 2011/0027256, Fusion Proteins; US 2010/0247509,Fusion Proteins; US 2010/0041098, Modified Clostridial Toxins withAltered Targeting Capabilities for Clostridial Toxin Target Cells; US2010/0034802, Treatment of Pain; US 2009/0162341, Non-Cytotoxic ProteinConjugates; US 2009/0087458, Activatable Recombinant Neurotoxins; US2009/0081730, Activatable Recombinant Neurotoxins; US 2009/0069238,Activatable Clostridial Toxins; US 2009/0042270, Activatable RecombinantNeurotoxins; US 2009/0030182, Activatable Recombinant Neurotoxins; US2009/0018081, Activatable Clostridial Toxins; US 2009/0005313,Activatable Clostridial Toxins; US 2009/0004224, Activatable ClostridialToxins; US 2008/0317783, Clostridial Toxin Derivatives and Methods forTreating Pain; US 2008/0241881, Modified Clostridial Toxins withEnhanced Translocation Capabilities and Altered Targeting Activity forClostridial Toxin Target Cells; WO 2006/099590, Modified ClostridialToxins with Altered Targeting Capabilities for Clostridial Toxin TargetCells; WO 2006/101809, Modified Clostridial Toxins with EnhancedTargeting Capabilities for Endogenous Clostridial Toxin ReceptorSystems; WO 2007/106115, Modified Clostridial Toxins with AlteredTargeting Capabilities for Clostridial Toxin Target Cells; WO2008/008803, Modified Clostridial Toxins with Enhanced TranslocationCapabilities and Altered Targeting Activity for Clostridial Toxin TargetCells; WO 2008/008805, Modified Clostridial Toxins with EnhancedTranslocation Capabilities and Altered Targeting Activity ForNon-Clostridial Toxin Target Cells; WO 2008/105901, Modified ClostridialToxins with Enhanced Translocation Capability and Enhanced TargetingActivity; WO 2011/020052, Methods of Treating Cancer Using OpioidRetargeted Endopeptidases; WO 2011/020056, Methods of Treating CancerUsing Galanin Retargeted Endopeptidases; WO 2011/020114, Methods ofTreating Cancer Using Tachykinin Retargeted Endopeptidases; WO2011/020115, Methods of Treating Cancer Using Growth Factor RetargetedEndopeptidases; WO 2011/020117, Methods of Treating Cancer UsingNeurotrophin Retargeted Endopeptidases; WO 2011/020119, Methods ofTreating Cancer Using Glucagon-Like Hormone Retargeted Endopeptidases;each incorporated entirely by reference.

Aspects of the present specification disclose, in part, a composition.In one aspect of this embodiment, a composition comprises a TEM asdisclosed herein. In another aspect of this embodiment, a compositioncomprises a Clostridial toxin and a TEM as disclosed herein. Any of thecompositions disclosed herein can be useful in a method of treatingdisclosed herein, with the proviso that the composition prevents orreduces a symptom associated with condition being treated. A Clostridialtoxin and a TEM as disclosed herein may be provided as separatecompositions or as part of a single composition. It is also understoodthat the two or more different Clostridial toxins and/or TEMs can beprovided as separate compositions or as part of a single composition.

A composition disclosed herein is generally administered as apharmaceutical acceptable composition. As used herein, the term“pharmaceutically acceptable” means any molecular entity or compositionthat does not produce an adverse, allergic or other untoward or unwantedreaction when administered to an individual. As used herein, the term“pharmaceutically acceptable composition” is synonymous with“pharmaceutical composition” and means a therapeutically effectiveconcentration of an active ingredient, such as, e.g., any of theClostridial toxins and/or TEMs disclosed herein. A pharmaceuticalcomposition disclosed herein is useful for medical and veterinaryapplications. A pharmaceutical composition may be administered to anindividual alone, or in combination with other supplementary activeingredients, agents, drugs or hormones. The pharmaceutical compositionsmay be manufactured using any of a variety of processes, including,without limitation, conventional mixing, dissolving, granulating,dragee-making, levigating, emulsifying, encapsulating, entrapping, andlyophilizing. The pharmaceutical composition can take any of a varietyof forms including, without limitation, a sterile solution, suspension,emulsion, lyophilizate, tablet, pill, pellet, capsule, powder, syrup,elixir or any other dosage form suitable for administration.

A pharmaceutical composition disclosed herein may optionally include apharmaceutically acceptable carrier that facilitates processing of anactive ingredient into pharmaceutically acceptable compositions. As usedherein, the term “pharmacologically acceptable carrier” is synonymouswith “pharmacological carrier” and means any carrier that hassubstantially no long term or permanent detrimental effect whenadministered and encompasses terms such as “pharmacologically acceptablevehicle, stabilizer, diluent, additive, auxiliary or excipient.” Such acarrier generally is mixed with an active ingredient, or permitted todilute or enclose the active compound and can be a solid, semi-solid, orliquid agent. It is understood that the active ingredients can besoluble or can be delivered as a suspension in the desired carrier ordiluent. Any of a variety of pharmaceutically acceptable carriers can beused including, without limitation, aqueous media such as, e.g., water,saline, glycine, hyaluronic acid and the like; solid carriers such as,e.g., mannitol, lactose, starch, magnesium stearate, sodium saccharin,talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like;solvents; dispersion media; coatings; antibacterial and antifungalagents; isotonic and absorption delaying agents; or any other inactiveingredient. Selection of a pharmacologically acceptable carrier candepend on the mode of administration. Except insofar as anypharmacologically acceptable carrier is incompatible with the activeingredient, its use in pharmaceutically acceptable compositions iscontemplated. Non-limiting examples of specific uses of suchpharmaceutical carriers can be found in PHARMACEUTICAL DOSAGE FORMS ANDDRUG D ELIVERY SYSTEMS (Howard C. Ansel et al., eds., LippincottWilliams & Wilkins Publishers, 7^(th) ed. 1999); REMINGTON: THE SCIENCEAND P RACTICE OF PHARMACY (Alfonso R. Gennaro ed., Lippincott, Williams& Wilkins, 20^(th) ed. 2000); GOODMAN & GILMAN'S THE P HARMACOLOGICAL BASIS OF T HERAPEUTICS (Joel G. Hardman et al., eds., McGraw-HillProfessional, 10^(th) ed. 2001); and HANDBOOK OF PHARMACEUTICALEXCIPIENTS (Raymond C. Rowe et al., APhA Publications, 4^(th) edition2003). These protocols are routine procedures and any modifications arewell within the scope of one skilled in the art and from the teachingherein.

A pharmaceutical composition disclosed herein can optionally include,without limitation, other pharmaceutically acceptable components (orpharmaceutical components), including, without limitation, buffers,preservatives, tonicity adjusters, salts, antioxidants, osmolalityadjusting agents, physiological substances, pharmacological substances,bulking agents, emulsifying agents, wetting agents, sweetening orflavoring agents, and the like. Various buffers and means for adjustingpH can be used to prepare a pharmaceutical composition disclosed herein,provided that the resulting preparation is pharmaceutically acceptable.Such buffers include, without limitation, acetate buffers, citratebuffers, phosphate buffers, neutral buffered saline, phosphate bufferedsaline and borate buffers. It is understood that acids or bases can beused to adjust the pH of a composition as needed. Pharmaceuticallyacceptable antioxidants include, without limitation, sodiummetabisulfite, sodium thiosulfate, acetylcysteine, butylatedhydroxyanisole and butylated hydroxytoluene. Useful preservativesinclude, without limitation, benzalkonium chloride, chlorobutanol,thimerosal, phenylmercuric acetate, phenylmercuric nitrate, a stabilizedoxy chloro composition and chelants, such as, e.g., DTPA orDTPA-bisamide, calcium DTPA, and CaNa-DTPA-bisamide. Tonicity adjustorsuseful in a pharmaceutical composition include, without limitation,salts such as, e.g., sodium chloride, potassium chloride, mannitol orglycerin and other pharmaceutically acceptable tonicity adjustor. Thepharmaceutical composition may be provided as a salt and can be formedwith many acids, including but not limited to, hydrochloric, sulfuric,acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be moresoluble in aqueous or other protonic solvents than are the correspondingfree base forms. It is understood that these and other substances knownin the art of pharmacology can be included in a pharmaceuticalcomposition. Exemplary pharmaceutical composition comprising a TEM aredescribed in Hunt, et al., Animal Protein-Free PharmaceuticalCompositions, U.S. Ser. No. 12/331,816; and Dasari, et al., ClostridialToxin Pharmaceutical Compositions, WO/2010/090677, each incorporatedentirely by reference.

In an embodiment, a composition is a pharmaceutical compositioncomprising a TEM. In aspects of this embodiment, a pharmaceuticalcomposition comprising a TEM further comprises a pharmacologicalcarrier, a pharmaceutical component, or both a pharmacological carrierand a pharmaceutical component. In other aspects of this embodiment, apharmaceutical composition comprising a TEM further comprises at leastone pharmacological carrier, at least one pharmaceutical component, orat least one pharmacological carrier and at least one pharmaceuticalcomponent.

In another embodiment, a composition is a pharmaceutical compositioncomprising a Clostridial toxin. In aspects of this embodiment, apharmaceutical composition comprising a Clostridial toxin furthercomprises a pharmacological carrier, a pharmaceutical component, or botha pharmacological carrier and a pharmaceutical component. In otheraspects of this embodiment, a pharmaceutical composition comprising aClostridial toxin further comprises at least one pharmacologicalcarrier, at least one pharmaceutical component, or at least onepharmacological carrier and at least one pharmaceutical component.

In yet another embodiment, a composition is a pharmaceutical compositioncomprising a Clostridial toxin and a TEM. In aspects of this embodiment,a pharmaceutical composition comprising a Clostridial toxin and a TEMfurther comprises a pharmacological carrier, a pharmaceutical component,or both a pharmacological carrier and a pharmaceutical component. Inother aspects of this embodiment, a pharmaceutical compositioncomprising a Clostridial toxin and a TEM further comprises at least onepharmacological carrier, at least one pharmaceutical component, or atleast one pharmacological carrier and at least one pharmaceuticalcomponent.

A post-traumatic stress disorder (PTSD) refers to a disorder where anindividual has one or more symptoms due to an over-reactive adrenalineresponse to a triggering traumatic event, which creates neurologicalpatterns in the brain and biochemical changes in the brain and body thatpersist long after the triggering event is over. As a result, anindividual affected with a PTSD is hyper-responsive to future fearful orstressful events. Some biochemical changes seen in PTSD sufferersinclude a low level of secretion of cortisol and a high level ofsecretion of catecholamines and corticotropin-releasing factor in theurine.

Symptoms of a PTSD can include, without limitation: amnesia regardingparts or all of the event; anger; avoidance of people, places or thingsthat may serve as reminders of the event; flashbacks, dreams or othermemories that cause the sufferer to re-experience the traumatic event;hypervigilance; insomnia; lack of concentration; emotional numbing of orcomplete inability to feel certain feelings; an intensely negativeresponse, either psychological or physiological or both, to reminders ofthe event; reduction in ability to participate in significant lifeactivities; and, a significant impairment of major areas of lifeactivity, such as social relations, work, etc. Additionally, alcohol anddrug abuse can commonly co-occur with a PTSD, and other psychologicaldisorders (such as an anxiety disorder) can be exacerbated or worsened).In some cases, a PTSD can become chronic.

A composition or compound is administered to an individual. Anindividual comprises all mammals, preferably a human being. Typically,any individual who is a candidate for a conventional psychologicaltrauma disorder treatment is a candidate for a psychological traumadisorder treatment disclosed herein. Pre-operative evaluation typicallyincludes routine history and physical examination in addition tothorough informed consent disclosing all relevant risks and benefits ofthe procedure.

With reference to a therapy comprising a TEM, the amount of a TEMdisclosed herein used with the methods of treatment disclosed hereinwill typically be an effective amount. As used herein, the term“effective amount” is synonymous with “therapeutically effectiveamount”, “effective dose”, or “therapeutically effective dose” and whenused in reference to treating a psychological trauma disorder means theminimum dose of a TEM alone necessary to achieve the desired therapeuticeffect and includes a dose sufficient to reduce a symptom associatedwith a psychological trauma disorder. An effective amount refers to thetotal amount of a TEM administered to an individual in one setting. Assuch, an effective amount of a TEM does not refer to the amountadministered per site. The effectiveness of a TEM disclosed herein intreating a psychological trauma disorder can be determined by observingan improvement in an individual based upon one or more clinicalsymptoms, and/or physiological indicators associated with the condition.An improvement in a psychological trauma disorder also can be indicatedby a reduced need for a concurrent therapy.

With reference to a standard dose combination therapy comprising aClostridial toxin and a TEM, an effective amount of a Clostridial toxinis one where in combination with a TEM the amount of a Clostridial toxinachieves the desired therapeutic effect. For example, typically about29-195 U of BOTOX® (Allergan, Inc., Irvine, Calif.), a BoNT/A, isadministered in order to treat a psychological trauma disorder. As oneexample, a patient may be treated according to the PREEMPT paradigm with155 U BoNT/A. In another case, a female patient may be treated using aglabellar administration paradigm with a total of about 30 U BoNT/A, amale patient treated using a glaballar administration paradigm with atotal of about 40 U BoNT/A.

Generally, the PREEMPT paradigm refers to administration to: thefrontalis at divided among four sites of administration; to thecorrugator divided among two sites of administration; to the procerus atone site of administration; to the occipitalis at six sites to eightsites of administration; to the temporalis at eight sites to ten sitesof administration; to the trapezius at six sites to ten sites ofadministration and to the cervical paraspinal muscles at four sites ofadministration. In one embodiment, a total of 155 units of BoNT/A areadministered at 31 sites.

With reference to a low dose combination therapy comprising aClostridial toxin and a TEM, an effective amount of a Clostridial toxinis one where in combination with a TEM the amount of a Clostridial toxinachieves the desired therapeutic effect, but such an amount administeredon its own would be ineffective. For example, typically about 29-195 Uof BOTOX® (Allergan, Inc., Irvine, Calif.), a BoNT/A, is administered inorder to treat a psychological trauma disorder. However, in a low dosecombination therapy, a suboptimal effective amount of BoNT/A would beadministered to treat a psychological trauma disorder when such toxin isused in a combined therapy with a TEM. For example, less that 50 U, lessthan 25 U, less than 15 U, less than 10 U, less than 7.5 U, less than 5U, less than 2.5 U, or less than 1 U of BoNT/A would be administered totreat a psychological trauma disorder when used in a low dosecombination therapy with a TEM as disclosed herein.

The appropriate effective amount of a Clostridial toxin and/or a TEM tobe administered to an individual for a particular psychological traumadisorder can be determined by a person of ordinary skill in the art bytaking into account factors, including, without limitation, the type ofpsychological trauma disorder, the location of the psychological traumadisorder, the cause of the psychological trauma disorder, the severityof the psychological trauma disorder, the degree of relief desired, theduration of relief desired, the particular Clostridial toxin and/or aTEM used, the rate of excretion of the Clostridial toxin and/or a TEMused, the pharmacodynamics of the Clostridial toxin and/or a TEM used,the nature of the other compounds to be included in the composition, theparticular route of administration, the particular characteristics,history and risk factors of the individual, such as, e.g., age, weight,general health and the like, or any combination thereof. Additionally,where repeated administration of a composition comprising disclosedherein is used, an effective amount of a Clostridial toxin and/or a TEMwill further depend upon factors, including, without limitation, thefrequency of administration, the half-life of the composition comprisinga Clostridial toxin and/or a TEM, or any combination thereof. In isknown by a person of ordinary skill in the art that an effective amountof a composition comprising a Clostridial toxin and/or a TEM can beextrapolated from in vitro assays and in vivo administration studiesusing animal models prior to administration to humans.

Wide variations in the necessary effective amount are to be expected inview of the differing efficiencies of the various routes ofadministration. For instance, oral administration generally would beexpected to require higher dosage levels than administration byintravenous or intravitreal injection. Similarly, systemicadministration of a TEM would be expected to require higher dosagelevels than a local administration. Variations in these dosage levelscan be adjusted using standard empirical routines of optimization, whichare well-known to a person of ordinary skill in the art. The precisetherapeutically effective dosage levels and patterns are preferablydetermined by the attending physician in consideration of theabove-identified factors. One skilled in the art will recognize that thecondition of the individual can be monitored throughout the course oftherapy and that the effective amount of a TEM disclosed herein that isadministered can be adjusted accordingly.

In aspects of this embodiment, a therapeutically effective amount of acomposition comprising a TEM reduces a symptom associated with apsychological trauma disorder by, e.g., at least 10%, at least 20%, atleast 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90% or at least 100%. In other aspects of thisembodiment, a therapeutically effective amount of a compositioncomprising a TEM reduces a symptom associated with a psychologicaltrauma disorder by, e.g., at most 10%, at most 20%, at most 30%, at most40%, at most 50%, at most 60%, at most 70%, at most 80%, at most 90% orat most 100%. In yet other aspects of this embodiment, a therapeuticallyeffective amount of a composition comprising a TEM reduces a symptomassociated with a psychological trauma disorder by, e.g., about 10% toabout 100%, about 10% to about 90%, about 10% to about 80%, about 10% toabout 70%, about 10% to about 60%, about 10% to about 50%, about 10% toabout 40%, about 20% to about 100%, about 20% to about 90%, about 20% toabout 80%, about 20% to about 20%, about 20% to about 60%, about 20% toabout 50%, about 20% to about 40%, about 30% to about 100%, about 30% toabout 90%, about 30% to about 80%, about 30% to about 70%, about 30% toabout 60%, or about 30% to about 50%. In still other aspects of thisembodiment, a therapeutically effective amount of the TEM is the dosagesufficient to inhibit neuronal activity for, e.g., at least one week, atleast one month, at least two months, at least three months, at leastfour months, at least five months, at least six months, at least sevenmonths, at least eight months, at least nine months, at least tenmonths, at least eleven months, or at least twelve months.

In other aspects of this embodiment, a therapeutically effective amountof a TEM generally is in the range of about 1 fg to about 3.0 mg. Inaspects of this embodiment, an effective amount of a TEM can be, e.g.,about 100 fg to about 3.0 mg, about 100 pg to about 3.0 mg, about 100 ngto about 3.0 mg, or about 100 pg to about 3.0 mg. In other aspects ofthis embodiment, an effective amount of a TEM can be, e.g., about 100 fgto about 750 μg, about 100 pg to about 750 μg, about 100 ng to about 750μg, or about 1 μg to about 750 μg. In yet other aspects of thisembodiment, a therapeutically effective amount of a TEM can be, e.g., atleast 1 fg, at least 250 fg, at least 500 fg, at least 750 fg, at least1 pg, at least 250 pg, at least 500 pg, at least 750 pg, at least 1 ng,at least 250 ng, at least 500 ng, at least 750 ng, at least 1 μg, atleast 250 μg, at least 500 μg, at least 750 μg, or at least 1 mg. Instill other aspects of this embodiment, a therapeutically effectiveamount of a composition comprising a TEM can be, e.g., at most 1 fg, atmost 250 fg, at most 500 fg, at most 750 fg, at most 1 pg, at most 250pg, at most 500 pg, at most 750 pg, at most 1 ng, at most 250 ng, atmost 500 ng, at most 750 ng, at most 1 μg, at least 250 μg, at most 500μg, at most 750 μg, or at most 1 mg.

In yet other aspects of this embodiment, a therapeutically effectiveamount of a TEM generally is in the range of about 0.00001 mg/kg toabout 3.0 mg/kg. In aspects of this embodiment, an effective amount of aTEM can be, e.g., about 0.0001 mg/kg to about 0.001 mg/kg, about 0.03mg/kg to about 3.0 mg/kg, about 0.1 mg/kg to about 3.0 mg/kg, or about0.3 mg/kg to about 3.0 mg/kg. In yet other aspects of this embodiment, atherapeutically effective amount of a TEM can be, e.g., at least 0.00001mg/kg, at least 0.0001 mg/kg, at least 0.001 mg/kg, at least 0.01 mg/kg,at least 0.1 mg/kg, or at least 1 mg/kg. In yet other aspects of thisembodiment, a therapeutically effective amount of a TEM can be, e.g., atmost 0.00001 mg/kg, at most 0.0001 mg/kg, at most 0.001 mg/kg, at most0.01 mg/kg, at most 0.1 mg/kg, or at most 1 mg/kg.

In aspects of this embodiment, a therapeutically effective amount of acomposition comprising a Clostridial toxin reduces a symptom associatedwith a psychological trauma disorder by, e.g., at least 10%, at least20%, at least 30%, at least 40%, at least 50%, at least 60%, at least70%, at least 80%, at least 90% or at least 100%. In other aspects ofthis embodiment, a therapeutically effective amount of a compositioncomprising a Clostridial toxin reduces a symptom associated with apsychological trauma disorder by, e.g., at most 10%, at most 20%, atmost 30%, at most 40%, at most 50%, at most 60%, at most 70%, at most80%, at most 90% or at most 100%. In yet other aspects of thisembodiment, a therapeutically effective amount of a compositioncomprising a Clostridial toxin reduces a symptom associated with apsychological trauma disorder by, e.g., about 10% to about 100%, about10% to about 90%, about 10% to about 80%, about 10% to about 70%, about10% to about 60%, about 10% to about 50%, about 10% to about 40%, about20% to about 100%, about 20% to about 90%, about 20% to about 80%, about20% to about 20%, about 20% to about 60%, about 20% to about 50%, about20% to about 40%, about 30% to about 100%, about 30% to about 90%, about30% to about 80%, about 30% to about 70%, about 30% to about 60%, orabout 30% to about 50%. In still other aspects of this embodiment, atherapeutically effective amount of a Clostridial toxin is the dosagesufficient to inhibit neuronal activity for, e.g., at least one week, atleast one month, at least two months, at least three months, at leastfour months, at least five months, at least six months, at least sevenmonths, at least eight months, at least nine months, at least tenmonths, at least eleven months, or at least twelve months.

In other aspects of this embodiment, a therapeutically effective amountof a Clostridial toxin generally is in the range of about 1 fg to about30.0 μg. In other aspects of this embodiment, a therapeuticallyeffective amount of a Clostridial toxin can be, e.g., at least 1.0 pg,at least 10 pg, at least 100 pg, at least 1.0 ng, at least 10 ng, atleast 100 ng, at least 1.0 pg, at least 10 pg, at least 100 pg, or atleast 1.0 mg. In still other aspects of this embodiment, atherapeutically effective amount of a Clostridial toxin can be, e.g., atmost 1.0 μg, at most 10 μg, at most 100 μg, at most 1.0 ng, at most 10ng, at most 100 ng, at most 1.0 μg, at most 10 μg, at most 100 μg, or atmost 1.0 mg. In still other aspects of this embodiment, atherapeutically effective amount of a Clostridial toxin can be, e.g.,about 1.0 pg to about 10 μg, about 10 pg to about 10 μg, about 100 pg toabout 10 μg, about 1.0 ng to about 10 μg, about 10 ng to about 10 μg, orabout 100 ng to about 10 μg. In still other aspects of this embodiment,a therapeutically effective amount of a Clostridial toxin can be from,e.g., about 1.0 pg to about 1.0 μg, about 10 pg to about 1.0 μg, about100 pg to about 1.0 μg, about 1.0 ng to about 1.0 μg, about 10 ng toabout 1.0 μg, or about 100 ng to about 1.0 μg. In other aspects of thisembodiment, a therapeutically effective amount of a Clostridial toxincan be from, e.g., about 1.0 pg to about 100 ng, about 10 pg to about100 ng, about 100 pg to about 100 ng, about 1.0 ng to about 100 ng, orabout 10 ng to about 100 ng.

In yet other aspects of this embodiment, a therapeutically effectiveamount of a Clostridial toxin generally is in the range of about 0.1 Uto about 2500 U. In other aspects of this embodiment, a therapeuticallyeffective amount of a Clostridial toxin can be, e.g., at least 1.0 U, atleast 10 U, at least 100 U, at least 250 U, at least 500 U, at least 750U, at least 1,000 U, at least 1,500 U, at least 2,000 U, or at least2,500 U. In still other aspects of this embodiment, a therapeuticallyeffective amount of a Clostridial toxin can be, e.g., at most 1.0 U, atmost 10 U, at most 100 U, at most 250 U, at most 500 U, at most 750 U,at most 1,000 U, at most 1,500 U, at most 2,000 U, or at most 2,500 U.In still other aspects of this embodiment, a therapeutically effectiveamount of a Clostridial toxin can be, e.g., about 1 U to about 2,000 U,about 10 U to about 2,000 U, about 50 U to about 2,000 U, about 100 U toabout 2,000 U, about 500 U to about 2,000 U, about 1,000 U to about2,000 U, about 1 U to about 1,000 U, about 10 U to about 1,000 U, about50 U to about 1,000 U, about 100 U to about 1,000 U, about 500 U toabout 1,000 U, about 1 U to about 500 U, about 10 U to about 500 U,about 50 U to about 500 U, about 100 U to about 500 U, about 1 U toabout 100 U, about 10 U to about 100 U, about 50 U to about 100 U, about0.1 U to about 1 U, about 0.1 U to about 5 U, about 0.1 U to about 10 U,about 0.1 U to about 15 U, about 0.1 U to about 20 U, about 0.1 U toabout 25 U.

In still other aspects of this embodiment, a therapeutically effectiveamount of a Clostridial toxin generally is in the range of about 0.0001U/kg to about 3,000 U/kg. In aspects of this embodiment, atherapeutically effective amount of a Clostridial toxin can be, e.g., atleast 0.001 U/kg, at least 0.01 U/kg, at least 0.1 U/kg, at least 1.0U/kg, at least 10 U/kg, at least 100 U/kg, or at least 1000 U/kg. Inother aspects of this embodiment, a therapeutically effective amount ofa Clostridial toxin can be, e.g., at most 0.001 U/kg, at most 0.01 U/kg,at most 0.1 U/kg, at most 1.0 U/kg, at most 10 U/kg, at most 100 U/kg,or at most 1000 U/kg. In yet other aspects of this embodiment, atherapeutically effective amount of a Clostridial toxin can be between,e.g., about 0.001 U/kg to about 1 U/kg, about 0.01 U/kg to about 1 U/kg,about 0.1 U/kg to about 1 U/kg, about 0.001 U/kg to about 10 U/kg, about0.01 U/kg to about 10 U/kg, about 0.1 U/kg to about 10 U/kg about 1 U/kgto about 10 U/kg, about 0.001 U/kg to about 100 U/kg, about 0.01 U/kg toabout 100 U/kg, about 0.1 U/kg to about 100 U/kg, about 1 U/kg to about100 U/kg, or about 10 U/kg to about 100 U/kg. As used herein, the term“unit” or “U” is refers to the LD₅₀ dose, which is defined as the amountof a Clostridial toxin disclosed herein that killed 50% of the miceinjected with the Clostridial toxin.

In aspects of this embodiment, a therapeutically effective amount of astandard or low combination therapy comprising a Clostridial toxin and aTEM reduces a symptom associated with a psychological trauma disorderby, e.g., at least 10%, at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90% or atleast 100%. In other aspects of this embodiment, a therapeuticallyeffective amount of a standard or low combination therapy comprising aClostridial toxin and a TEM reduces a symptom associated with apsychological trauma disorder by, e.g., at most 10%, at most 20%, atmost 30%, at most 40%, at most 50%, at most 60%, at most 70%, at most80%, at most 90% or at most 100%. In yet other aspects of thisembodiment, a therapeutically effective amount of a standard or lowcombination therapy comprising a Clostridial toxin and a TEM reduces asymptom associated with a psychological trauma disorder by, e.g., about10% to about 100%, about 10% to about 90%, about 10% to about 80%, about10% to about 70%, about 10% to about 60%, about 10% to about 50%, about10% to about 40%, about 20% to about 100%, about 20% to about 90%, about20% to about 80%, about 20% to about 20%, about 20% to about 60%, about20% to about 50%, about 20% to about 40%, about 30% to about 100%, about30% to about 90%, about 30% to about 80%, about 30% to about 70%, about30% to about 60%, or about 30% to about 50%. In still other aspects ofthis embodiment, a therapeutically effective amount of a standard or lowcombination therapy comprising a Clostridial toxin and a TEM is thedosage sufficient to inhibit neuronal activity for, e.g., at least oneweek, at least one month, at least two months, at least three months, atleast four months, at least five months, at least six months, at leastseven months, at least eight months, at least nine months, at least tenmonths, at least eleven months, or at least twelve months.

In other aspects of this embodiment, a therapeutically effective amountof a standard or low combination therapy comprising a Botulinum toxinand a TEM generally is in a botulinum toxin: TEM molar ratio of about1:1 to about 1:10,000. In other aspects of this embodiment, atherapeutically effective amount of a standard or low combinationtherapy comprising a botulinum toxin and a TEM can be in a botulinumtoxin: TEM molar ratio of, e.g., about 1:1, about 1:2, about 1:5, about1:10, about 1:25, about 1:50, about 1:75, about 1:100, about 1:200,about 1:300, about 1:400, about 1:500, about 1:600, about 1:700, about1:800, about 1:900, about 1:1000, about 1:2000, about 1:3000, about1:4000, about 1:5000, about 1:6000, about 1:7000, about 1:8000, about1:9000, or about 1:10,000. In yet other aspects of this embodiment, atherapeutically effective amount of standard or low combination therapycomprising a botulinum toxin and a TEM can be in a botulinum toxin: TEMmolar ratio of, e.g., at least 1:1, at least 1:2, at least 1:5, at least1:10, at least 1:25, at least 1:50, at least 1:75, at least 1:100, atleast 1:200, at least 1:300, at least 1:400, at least 1:500, at least1:600, at least 1:700, at least 1:800, at least 1:900, at least 1:1000,at least 1:2000, at least 1:3000, at least 1:4000, at least 1:5000, atleast 1:6000, at least 1:7000, at least 1:8000, at least 1:9000, or atleast 1:10,000. In still other aspects of this embodiment, atherapeutically effective amount of a standard or low combinationtherapy comprising a botulinum toxin and a TEM can be in a botulinumtoxin: TEM molar ratio of between, e.g., about 1:1 to about 1:10,000,about 1:10 to about 1:10,000, about 1:100 to about 1:10,000, about 1:500to about 1:10,000, about 1:1000 to about 1:10,000, about 1:5000 to about1:10,000, about 1:1 to about 1:1000, about 1:10 to about 1:1000, about1:100 to about 1:1000, about 1:250 to about 1:1000, about 1:500 to about1:1000, about 1:750 to about 1:1000, about 1:1 to about 1:500, about1:10 to about 1:500, about 1:50 to about 1:500, about 1:100 to about1:500, about 1:250 to about 1:500, about 1:1 to about 1:100, about 1:10to about 1:100, about 1:25 to about 1:100, about 1:50 to about 1:100, orabout 1:75 to about 1:100.

In yet other aspects of this embodiment, a therapeutically effectiveamount of a standard combination therapy comprising a botulinum toxinand a TEM generally is in a range of about 0.50 U to about 250 U ofbotulinum toxin and about 0.1 μg to about 2,000.0 μg of a TEM. Inaspects of this embodiment, a therapeutically effective amount of acombined therapy comprising a botulinum toxin and a TEM can be, e.g.,about 0.1 U to about 10 U of a botulinum toxin and about 10 μg to about1,000 μg of a TEM, about 0.1 U to about 10 U of a botulinum toxin andabout 10 μg to about 500 μg of a TEM, about 0.1 U to about 10 U of abotulinum toxin and about 10 μg to about 100 μg of a TEM, about 0.5 U toabout 10 U of a botulinum toxin and about 10 μg to about 1,000 μg of aTEM, about 0.5 U to about 10 U of a botulinum toxin and about 10 μg toabout 500 μg of a TEM, about 0.5 U to about 10 U of a botulinum toxinand about 10 μg to about 100 μg of a TEM, about 1 U to about 10 U of abotulinum toxin and about 100 μg to about 1,000 μg of a TEM, about 1 Uto about 10 U of a botulinum toxin and about 100 μg to about 500 μg of aTEM, or about 1 U to about 10 U of a botulinum toxin and about 100 μg toabout 100 μg of a TEM.

In yet other aspects of this embodiment, a therapeutically effectiveamount of a low combination therapy comprising a botulinum toxin and aTEM generally is in a range of about 0.01 U to about 50 U of botulinumtoxin and about 0.1 μg to about 2,000.0 μg of a TEM. In aspects of thisembodiment, a therapeutically effective amount of a combined therapycomprising a botulinum toxin and a TEM can be, e.g., about 0.1 U toabout 10 U of a botulinum toxin and about 10 μg to about 1,000 μg of aTEM, about 0.1 U to about 10 U of a botulinum toxin and about 10 μg toabout 500 μg of a TEM, about 0.1 U to about 10 U of a botulinum toxinand about 10 μg to about 100 μg of a TEM, about 0.5 U to about 10 U of abotulinum toxin and about 10 μg to about 1,000 μg of a TEM, about 0.5 Uto about 10 U of a botulinum toxin and about 10 μg to about 500 μg of aTEM, about 0.5 U to about 10 U of a botulinum toxin and about 10 μg toabout 100 μg of a TEM, about 1 U to about 10 U of a botulinum toxin andabout 100 μg to about 1,000 pg of a TEM, about 1 U to about 10 U of abotulinum toxin and about 100 μg to about 500 μg of a TEM, or about 1 Uto about 10 U of a botulinum toxin and about 100 μg to about 100 μg of aTEM.

Dosing can be single dosage or cumulative (serial dosing), and can bereadily determined by one skilled in the art. For instance, treatment ofa psychological trauma disorder may comprise a one-time administrationof an effective dose of a composition disclosed herein. As anon-limiting example, an effective dose of a composition disclosedherein can be administered once to an individual, e.g., as a singleinjection or deposition at or near the site exhibiting a symptom of apsychological trauma disorder. Alternatively, treatment of apsychological trauma disorder may comprise multiple administrations ofan effective dose of a composition disclosed herein carried out over arange of time periods, such as, e.g., daily, once every few days,weekly, monthly or yearly. As a non-limiting example, a compositiondisclosed herein can be administered once or twice yearly to anindividual. The timing of administration can vary from individual toindividual, depending upon such factors as the severity of anindividual's symptoms. For example, an effective dose of a compositiondisclosed herein can be administered to an individual once a month foran indefinite period of time, or until the individual no longer requirestherapy. A person of ordinary skill in the art will recognize that thecondition of the individual can be monitored throughout the course oftreatment and that the effective amount of a composition disclosedherein that is administered can be adjusted accordingly.

A composition disclosed herein can be administered to an individualusing a variety of routes. Routes of administration suitable for amethod of treating a psychological trauma disorder as disclosed hereininclude both local and systemic administration. Local administrationresults in significantly more delivery of a composition to a specificlocation as compared to the entire body of the individual, whereas,systemic administration results in delivery of a composition toessentially the entire body of the individual. Routes of administrationsuitable for a method of treating a psychological trauma disorder asdisclosed herein also include both central and peripheraladministration. Central administration results in delivery of acomposition to essentially the central nervous system of an individualand includes, e.g., intrathecal administration, epidural administrationas well as a cranial injection or implant. Peripheral administrationresults in delivery of a composition to essentially any area of anindividual outside of the central nervous system and encompasses anyroute of administration other than direct administration to the spine orbrain. The actual route of administration of a composition disclosedherein used can be determined by a person of ordinary skill in the artby taking into account factors, including, without limitation, the typeof psychological trauma disorder, the location of the psychologicaltrauma disorder, the cause of the psychological trauma disorder, theseverity of the psychological trauma disorder, the degree of reliefdesired, the duration of relief desired, the particular botulinum toxinand/or TEM used, the rate of excretion of the botulinum toxin and/or TEMused, the pharmacodynamics of the botulinum toxin and/or TEM used, thenature of the other compounds to be included in the composition, theparticular route of administration, the particular characteristics,history and risk factors of the individual, such as, e.g., age, weight,general health and the like, or any combination thereof.

In an embodiment, a composition disclosed herein is administeredsystemically to an individual. In another embodiment, a compositiondisclosed herein is administered locally to an individual. In an aspectof this embodiment, a composition disclosed herein is administered to anerve of an individual. In another aspect of this embodiment, acomposition disclosed herein is administered to the area surrounding anerve of an individual.

A composition disclosed herein can be administered to an individualusing a variety of delivery mechanisms. The actual delivery mechanismused to administer a composition disclosed herein to an individual canbe determined by a person of ordinary skill in the art by taking intoaccount factors, including, without limitation, the type ofpsychological trauma disorder, the location of the psychological traumadisorder, the cause of the psychological trauma disorder, the severityof the psychological trauma disorder, the degree of relief desired, theduration of relief desired, the particular botulinum toxin and/or TEMused, the rate of excretion of the botulinum toxin and/or TEM used, thepharmacodynamics of the botulinum toxin and/or TEM used, the nature ofthe other compounds to be included in the composition, the particularroute of administration, the particular characteristics, history andrisk factors of the individual, such as, e.g., age, weight, generalhealth and the like, or any combination thereof.

In an embodiment, a composition disclosed herein is administered byinjection. In aspects of this embodiment, administration of acomposition disclosed herein is by, e.g., intramuscular injection,intraorgan injection, subdermal injection, dermal injection,intracranial injection, spinal injection, or injection into any otherbody area for the effective administration of a composition disclosedherein. In aspects of this embodiment, injection of a compositiondisclosed herein is to a nerve or into the area surrounding a nerve.

In another embodiment, a composition disclosed herein is administered bycatheter. In aspects of this embodiment, administration of a compositiondisclosed herein is by, e.g., a catheter placed in an epidural space.

A composition disclosed herein as disclosed herein can also beadministered to an individual in combination with other therapeuticcompounds to increase the overall therapeutic effect of the treatment.The use of multiple compounds to treat an indication can increase thebeneficial effects while reducing the presence of side effects.

Aspects of the present invention can also be described as follows:

-   1. A method of treating a psychological trauma disorder in an    individual, the method comprising the step of administering to the    individual in need thereof a therapeutically effective amount of a    composition including a BoNT/A and/or a TEM, wherein administration    of the composition reduces a symptom of the psychological trauma    disorder, thereby treating the individual.-   2. A use of a BoNT/A and/or a TEM in the manufacturing a medicament    for treating a psychological trauma disorder in an individual in    need thereof.-   3. A use of a BoNT/A and/or a TEM in the treatment of a    psychological trauma disorder in an individual in need thereof.-   4. A method of treating a psychological trauma disorder in an    individual, the method comprising the step of administering to the    individual in need thereof a therapeutically effective amount of a    composition including a botulinum neurotoxin and a TEM, wherein    administration of the composition reduces a symptom of the    psychological trauma disorder, thereby treating the individual.-   5. A use of a botulinum neurotoxin and a TEM in the manufacturing a    medicament for treating a psychological trauma disorder in an    individual in need thereof.-   6. A use of a botulinum neurotoxin and a TEM in the treatment of a    psychological trauma disorder in an individual in need thereof.-   7. The embodiments of 1 to 6, wherein the TEM comprises a linear    amino-to-carboxyl single polypeptide order of 1) a Clostridial toxin    enzymatic domain, a Clostridial toxin translocation domain, a    targeting domain, 2) a Clostridial toxin enzymatic domain, a    targeting domain, a Clostridial toxin translocation domain, 3) a    targeting domain, a Clostridial toxin translocation domain, and a    Clostridial toxin enzymatic domain, 4) a targeting domain, a    Clostridial toxin enzymatic domain, a Clostridial toxin    translocation domain, 5) a Clostridial toxin translocation domain, a    Clostridial toxin enzymatic domain and a targeting domain, or 6) a    Clostridial toxin translocation domain, a targeting domain and a    Clostridial toxin enzymatic domain.-   8. The embodiments of 1 to 6, wherein the TEM comprises a linear    amino-to-carboxyl single polypeptide order of 1) a Clostridial toxin    enzymatic domain, an exogenous protease cleavage site, a Clostridial    toxin translocation domain, a targeting domain, 2) a Clostridial    toxin enzymatic domain, an exogenous protease cleavage site, a    targeting domain, a Clostridial toxin translocation domain, 3) a    targeting domain, a Clostridial toxin translocation domain, an    exogenous protease cleavage site and a Clostridial toxin enzymatic    domain, 4) a targeting domain, a Clostridial toxin enzymatic domain,    an exogenous protease cleavage site, a Clostridial toxin    translocation domain, 5) a Clostridial toxin translocation domain,    an exogenous protease cleavage site, a Clostridial toxin enzymatic    domain and a targeting domain, or 6) a Clostridial toxin    translocation domain, an exogenous protease cleavage site, a    targeting domain and a Clostridial toxin enzymatic domain.-   9. The embodiments of 1 to 8, wherein the Clostridial toxin    translocation domain is a BoNT/A translocation domain, a BoNT/B    translocation domain, a BoNT/C1 translocation domain, a BoNT/D    translocation domain, a BoNT/E translocation domain, a BoNT/F    translocation domain, a BoNT/G translocation domain, a TeNT    translocation domain, a BaNT translocation domain, or a BuNT    translocation domain.-   10. The embodiments of 1 to 9, wherein the Clostridial toxin    enzymatic domain is a BoNT/A enzymatic domain, a BoNT/B enzymatic    domain, a BoNT/C1 enzymatic domain, a BoNT/D enzymatic domain, a    BoNT/E enzymatic domain, a BoNT/F enzymatic domain, a BoNT/G    enzymatic domain, a TeNT enzymatic domain, a BaNT enzymatic domain,    or a BuNT enzymatic domain.-   11. The embodiments of 1 to 10, wherein the targeting domain is a    sensory neuron targeting domain, a sympathetic neuron targeting    domain, or a parasympathetic neuron targeting domain.-   12. The embodiments of 1 to 10, wherein the targeting domain is an    opioid peptide targeting domain, a galanin peptide targeting domain,    a PAR peptide targeting domain, a somatostatin peptide targeting    domain, a neurotensin peptide targeting domain, a SLURP peptide    targeting domain, an angiotensin peptide targeting domain, a    tachykinin peptide targeting domain, a Neuropeptide Y related    peptide targeting domain, a kinin peptide targeting domain, a    melanocortin peptide targeting domain, or a granin peptide targeting    domain, a glucagon like hormone peptide targeting domain, a secretin    peptide targeting domain, a pituitary adenylate cyclase activating    peptide (PACAP) peptide targeting domain, a growth hormone-releasing    hormone (GHRH) peptide targeting domain, a vasoactive intestinal    peptide (VIP) peptide targeting domain, a gastric inhibitory peptide    (GIP) peptide targeting domain, a calcitonin peptide targeting    domain, a visceral gut peptide targeting domain, a neurotrophin    peptide targeting domain, a head activator (HA) peptide, a glial    cell line-derived neurotrophic factor (GDNF) family of ligands (GFL)    peptide targeting domain, a RF-amide related peptide (RFRP) peptide    targeting domain, a neurohormone peptide targeting domain, or a    neuroregulatory cytokine peptide targeting domain, an interleukin    (IL) targeting domain, vascular endothelial growth factor (VEGF)    targeting domain, an insulin-like growth factor (IGF) targeting    domain, an epidermal growth factor (EGF) targeting domain, a    Transformation Growth Factor-β (TGF-β) targeting domain, a Bone    Morphogenetic Protein (BMP) targeting domain, a Growth and    Differentiation Factor (GDF) targeting domain, an activin targeting    domain, or a Fibroblast Growth Factor (FGF) targeting domain, or a    Platelet-Derived Growth Factor (PDGF) targeting domain.-   13. The embodiments of 8 to 12, wherein the exogenous protease    cleavage site is a plant papain cleavage site, an insect papain    cleavage site, a crustacean papain cleavage site, an enterokinase    cleavage site, a human rhinovirus 3C protease cleavage site, a human    enterovirus 3C protease cleavage site, a tobacco etch virus protease    cleavage site, a Tobacco Vein Mottling Virus cleavage site, a    subtilisin cleavage site, a hydroxylamine cleavage site, or a    Caspase 3 cleavage site.-   14. The embodiments of 1 to 13, wherein the Clostridial neurotoxin    is a BoNT/A, a BoNT/B, a BoNT/C1, a BoNT/D, a BoNT/E, a BoNT/F, a    BoNT/G, a TeNT, a BaNT, a BuNT, or any combination thereof.-   15. The embodiments of 1 to 14, wherein the BoNT/A and/or a TEM is    administered to an Arnold's nerve or a nerve from the recurrent    laryngeal nerve complex.-   16. The embodiments of 1 to 14, wherein BoNT/A and/or a TEM is    administered using the PREEMPT paradigm.-   17. The embodiments of 1 to 14, wherein BoNT/A and/or a TEM is    administered to the glabellar complex.

EXAMPLES

The following non-limiting examples are provided for illustrativepurposes only in order to facilitate a more complete understanding ofrepresentative embodiments now contemplated. These examples should notbe construed to limit any of the embodiments described in the presentspecification, including those pertaining to the compounds,compositions, methods or uses of treating a psychological traumadisorder.

Example 1

A patient complains if being incredibly fearful and nervous when sheknows she will be having to deal with a future stressful event. Afterroutine history and physical examination, a physician diagnosis thepatient with a post-traumatic stress disorder involving abnormalsympathetic neuron activity that results in abnormal release ofepinehrine and noradrenaline, and identifies the nerves and/or musclesinvolved in the condition. The woman is treated by administration of acomposition comprising a BoNT/A as disclosed in the presentspecification, following the glabellar administration paradigm with 30U. Alternatively, the woman is treated by administration of acomposition comprising a TEM with a therapeutically effective amount.Alternatively, the woman may be treated by administering a compositioncomprising a TEM and a BoNT/A at 10 U as disclosed in the presentspecification. The patient's condition is monitored and after about 1week from treatment, the woman indicates she has decreased fear andnervousness associated with an upcoming stressful event since thetreatment. At two and four month check-ups, the woman indicates that sheis still experiencing decreased fear and nervousness for upcomingfearful or stressful events. This decrease in fear and nervousness forupcoming fearful or stressful events indicates a successful treatmentwith the composition comprising a TEM and a BoNT/A as disclosed in thepresent specification.

A similar therapeutic effect can be achieved with a suboptimal amount ofany of the Clostridial toxins disclosed herein.

Example 2

A patient complains having a difficult time falling and staying asleep,being more irritable and having outbursts of anger, having difficultyconcentrating, feeling constantly “on guard” like danger is lurkingaround every corner, being “jumpy,” having a difficult timeconcentrating, having “flashbacks” to combat, and feeling as log hislife will be cut short.

The man is thoroughly examined by a doctor and diagnosed with posttraumatic stress disorder. The doctor concludes that botulinum toxintherapy is appropriate. A total of 100 Units of Botox® is administeredto the sphenopalatine ganglion nerves, with an additional 100 Unitsadministered to the face, head and neck. Alternatively, the man istreated by administration of a therapeutically effective amount of acomposition comprising a TEM. Alternatively, the man may be treated byadministering a composition comprising a TEM and a decreased amount ofBoNT/A relative to BoNT/A alone. Within days, the patient reports adecrease in his symptoms. The patient is evaluated at three months todetermine whether another round of administrations is necessary.

Example 3

A former soldier complains having a difficult time falling and stayingasleep, being more irritable and having outbursts of anger, havingdifficulty concentrating, feeling constantly “on guard” like danger islurking around every corner, being “jumpy,” having a difficult timeconcentrating, having “flashbacks” to combat, and feeling as log hislife will be cut short.

The man is thoroughly examined by a doctor and diagnosed with posttraumatic stress disorder. The doctor concludes that a combination ofbotulinum toxin therapy and TEM therapy is appropriate. A total of 100Units of Botox® is administered to the sphenopalatine ganglion nerves,with an additional 100 Units administered to the face, head and neck.Within days, the patient reports a decrease in his symptoms. The patientis evaluated at three months to determine whether another round ofadministrations is necessary.

Example 4

A student is a assaulted while walking back to her dorm room from thecampus library. The woman complains of a markedly diminished interest insignificant activities; a feeling detachment and estrangement fromothers in her life; she has a sense of a foreshortened future; avoidanceof the library; fear of walking alone even during the day; difficultyconcentrating; trouble sleeping and nightmares.

The woman is thoroughly interviewed and examined by a doctor and isdiagnosed with traumatic stress disorder. The doctor concludes thatbotulinum toxin therapy is appropriate. Botox® is administered to 31sites in the head, neck and shoulders. Specifically, 155 units of Botox®is administered to: the frontalis at about twenty units divided amongfour sites of administration; to the corrugator at about ten unitsdivided among two sites of administration; to the procerus at about fiveunits to one site of administration; to the occipitalis at about thirtyunits divided among six sites of administration to about forty unitsdivided among eight sites of administration; to the temporalis at aboutforty units divided among eight sites of administration up to fiftyunits divided among ten sites of administration; to the trapezius atabout thirty units divided among six sites of administration up to aboutfifty units divided among ten sites of administration and to thecervical paraspinal muscles at about twenty units divided among foursites of administration. Within days, the patient reports a decrease inher symptoms. The patient is evaluated at three months to determinewhether another round of administrations is necessary.

Example 5

A student is a assaulted while walking back to her dorm room from thecampus library. The woman complains of a markedly diminished interest insignificant activities; a feeling detachment and estrangement fromothers in her life; she has a sense of a foreshortened future; avoidanceof the library; fear of walking alone even during the day; difficultyconcentrating; trouble sleeping and nightmares.

The woman is thoroughly interviewed and examined by a doctor and isdiagnosed with traumatic stress disorder. The doctor concludes that acombination botulinum toxin therapy with TEM therapy is appropriate.Botox® and a TEM are co-administered to 31 sites in the head, neck andshoulders. Specifically, 50 units of Botox® and a 30-fold molar ratio ofa TEM are administered to: the frontalis at divided among four sites ofadministration; to the corrugator divided among two sites ofadministration; to the procerus at one site of administration; to theoccipitalis at six sites to eight sites of administration; to thetemporalis at eight sites to ten sites of administration; to thetrapezius at six sites to ten sites of administration and to thecervical paraspinal muscles at four sites of administration. Withindays, the patient reports a decrease in her symptoms. The patient isevaluated at three months to determine whether another round ofadministrations is necessary.

Example 6

A woman is caught in a severe storm while visiting relatives in Georgia.She survives a tornado by hiding in a closet on the ground floor, butmost of the house is destroyed and the next morning she learns that manyneighbors were killed. Two months after returning home, the womancomplains of a feeling detachment and estrangement from others in herlife; a sense of a foreshortened future; difficulty concentrating;trouble sleeping and nightmares. She also develops a strong fear ofrain, thunder, and loud winds.

The woman is thoroughly interviewed and examined by a doctor and isdiagnosed with traumatic stress disorder. The doctor concludes thatbotulinum toxin therapy is appropriate. Botulinum toxin is administeredto sites on the face and in the head, neck and shoulders. Atherapeutically effective amount of botulinum toxin is administered tothe sphenopalatine ganglion nerves, with an additional amountadministered to the face, head and neck. Within days, the patientreports a decrease in his symptoms. The patient is evaluated at threemonths to determine whether another round of administrations isnecessary. Within days, the patient reports a decrease in her symptoms.The patient is evaluated at three months to determine whether anotherround of administrations is necessary.

Example 7

A dog is caught in a severe storm and survives a tornado by hiding in acloset with his owner. Two month later, the dog is constantly agitated,vomits regularly, and curls up and whimpers at even moderate sounds. Thedog is examined by a veterinarian who determines the dog is sufferingfrom PTSD. The veterinarian administers a therapeutically effectiveamount of BoNT/A to the glabellar complex. Alternatively, theveterinarian could administer a TEM or a combination of BoNT/A and TEM.One week later, the PTSD symptoms are decreased. The dog is evaluatedthree months later to determine if more treatment is necessary.Alternatively, the veterinarian could administer according to a PREEMPTparadigm.

In closing, it is to be understood that although aspects of the presentspecification are highlighted by referring to specific embodiments, oneskilled in the art will readily appreciate that these disclosedembodiments are only illustrative of the principles of the subjectmatter disclosed herein. Therefore, it should be understood that thedisclosed subject matter is in no way limited to a particularmethodology, protocol, and/or reagent, etc., described herein. As such,various modifications or changes to or alternative configurations of thedisclosed subject matter can be made in accordance with the teachingsherein without departing from the spirit of the present specification.Lastly, the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to limit the scope ofthe present invention, which is defined solely by the claims.Accordingly, the present invention is not limited to that precisely asshown and described.

Certain embodiments of the present invention are described herein,including the best mode known to the inventors for carrying out theinvention. Of course, variations on these described embodiments willbecome apparent to those of ordinary skill in the art upon reading theforegoing description. The inventor expects skilled artisans to employsuch variations as appropriate, and the inventors intend for the presentinvention to be practiced otherwise than specifically described herein.Accordingly, this invention includes all modifications and equivalentsof the subject matter recited in the claims appended hereto as permittedby applicable law. Moreover, any combination of the above-describedembodiments in all possible variations thereof is encompassed by theinvention unless otherwise indicated herein or otherwise clearlycontradicted by context.

Groupings of alternative embodiments, elements, or steps of the presentinvention are not to be construed as limitations. Each group member maybe referred to and claimed individually or in any combination with othergroup members disclosed herein. It is anticipated that one or moremembers of a group may be included in, or deleted from, a group forreasons of convenience and/or patentability. When any such inclusion ordeletion occurs, the specification is deemed to contain the group asmodified thus fulfilling the written description of all Markush groupsused in the appended claims.

Unless otherwise indicated, all numbers expressing a characteristic,item, quantity, parameter, property, term, and so forth used in thepresent specification and claims are to be understood as being modifiedin all instances by the term “about.” As used herein, the term “about”means that the characteristic, item, quantity, parameter, property, orterm so qualified encompasses a range of plus or minus ten percent aboveand below the value of the stated characteristic, item, quantity,parameter, property, or term. Accordingly, unless indicated to thecontrary, the numerical parameters set forth in the specification andattached claims are approximations that may vary. At the very least, andnot as an attempt to limit the application of the doctrine ofequivalents to the scope of the claims, each numerical indication shouldat least be construed in light of the number of reported significantdigits and by applying ordinary rounding techniques. Notwithstandingthat the numerical ranges and values setting forth the broad scope ofthe invention are approximations, the numerical ranges and values setforth in the specific examples are reported as precisely as possible.Any numerical range or value, however, inherently contains certainerrors necessarily resulting from the standard deviation found in theirrespective testing measurements. Recitation of numerical ranges ofvalues herein is merely intended to serve as a shorthand method ofreferring individually to each separate numerical value falling withinthe range. Unless otherwise indicated herein, each individual value of anumerical range is incorporated into the present specification as if itwere individually recited herein.

The terms “a,” “an,” “the” and similar referents used in the context ofdescribing the present invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. All methods described herein can be performed in any suitableorder unless otherwise indicated herein or otherwise clearlycontradicted by context. The use of any and all examples, or exemplarylanguage (e.g., “such as”) provided herein is intended merely to betterilluminate the present invention and does not pose a limitation on thescope of the invention otherwise claimed. No language in the presentspecification should be construed as indicating any non-claimed elementessential to the practice of the invention.

Specific embodiments disclosed herein may be further limited in theclaims using consisting of or consisting essentially of language. Whenused in the claims, whether as filed or added per amendment, thetransition term “consisting of” excludes any element, step, oringredient not specified in the claims. The transition term “consistingessentially of” limits the scope of a claim to the specified materialsor steps and those that do not materially affect the basic and novelcharacteristic(s). Embodiments of the present invention so claimed areinherently or expressly described and enabled herein.

All patents, patent publications, and other publications referenced andidentified in the present specification are individually and expresslyincorporated herein by reference in their entirety for the purpose ofdescribing and disclosing, for example, the compositions andmethodologies described in such publications that might be used inconnection with the present invention. These publications are providedsolely for their disclosure prior to the filing date of the presentapplication. Nothing in this regard should be construed as an admissionthat the inventors are not entitled to antedate such disclosure byvirtue of prior invention or for any other reason. All statements as tothe date or representation as to the contents of these documents isbased on the information available to the applicants and does notconstitute any admission as to the correctness of the dates or contentsof these documents.

We claim:
 1. A method of treating at least one symptom of post-traumaticstress disorder in a patient in need thereof, the method comprising thestep of administering to the patient a therapeutically effective amountof a composition including a botulinum neurotoxin (BoNT), whereinadministration of the composition decreases the at least one symptom ofthe post-traumatic stress disorder; and wherein the at least one symptomof post-traumatic stress disorder comprises total or partial amnesia ofa traumatic event, flashbacks or nightmares wherein the patient re-experiences the traumatic event, avoidance of stimuli associated withthe traumatic event, increased arousal including difficulty falling orstaying asleep, anger, hyper-vigilance, or combinations thereof.
 2. Themethod of claim 1, wherein the BoNT is administered to an Arnold's nerveor a nerve from the recurrent laryngeal nerve complex.
 3. The method ofclaim 1, wherein the BoNT is administered to: the frontalis at dividedamong four sites of administration; to the corrugator divided among twosites of administration; to the procerus at one site of administration;to the occipitalis at six sites to eight sites of administration; to thetemporalis at eight sites to ten sites of administration; to thetrapezius at six sites to ten sites of administration and to thecervical paraspinal muscles at four sites of administration.
 4. Themethod of claim 1, wherein the BoNT is administered to the glabellarcomplex.
 5. The method of claim 1, wherein the BoNT is BoNT/A.
 6. Amethod of treating at least one symptom of a psychological traumadisorder in a patient in need thereof, the method comprising the step ofadministering to the mammal in need thereof a therapeutically effectiveamount of a composition including a botulinum neurotoxin (BoNT) and/or aTargeted Exocytosis Modulator (TEM), wherein administration of thecomposition reduces the at least one symptom of the psychological traumadisorder, thereby treating the patient.
 7. The method of claim 6,wherein the psychological trauma disorder is a post-traumatic stressdisorder.
 8. The method of claim 6, wherein the BoNT and/or TEM areadministered to an Arnold's nerve or a nerve from the recurrentlaryngeal nerve complex.
 9. The method of claim 6, wherein the BoNTand/or TEM are administered to: the frontalis at divided among foursites of administration; to the corrugator divided among two sites ofadministration; to the procerus at one site of administration; to theoccipitalis at six sites to eight sites of administration; to thetemporalis at eight sites to ten sites of administration; to thetrapezius at six sites to ten sites of administration and to thecervical paraspinal muscles at four sites of administration.
 10. Themethod of claim 6, wherein the BoNT and/or TEM are administered to theglabellar complex.
 11. The method of claim 6, wherein the BoNT isBoNT/A.